Coleopteran-resistant transgenic plants and methods of their production

ABSTRACT

Disclosed are nucleic acid segments comprising synthetically-modified genes encoding Coleopteran-toxic  B. thuringiensis  δ-endotoxins. Also disclosed are methods of using these genes for the recombinant expression of polypeptides, the preparation of vectors containing the genes, and methods for transforming suitable host cells.

This application is a divisional of application Ser. No. 09/427,770filed Oct. 27, 1999 now U.S. Pat. No. 6,620,988, which is a continuationof Ser. No. 08/993,722, filed Dec. 18, 1997, now U.S. Pat. No.6,060,594.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

This invention relates to transformed host cells and vectors whichcomprise nucleic acid segments encoding genetically-engineered,recombinant Bacillus thuringiensis δ-endotoxins which are active againstColeopteran insects.

1.2 Description of the Related Art

Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are Coleopteran and Lepidopteran pests. For example,vegetable and cole crops such as artichokes, kohirabi, arugula, leeks,asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets,bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon,crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni,carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas,parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes,cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga,eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach,green onions, squash, greens, sugar beets, sweet potatoes, turnip, swisschard, horseradish, tomatoes, kale, turnips, and a variety of spices aresensitive to infestation by one or more of the following insect pests:alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbagebudworm, cabbage looper, cabbage webworm, corn earworm, celeryleafeater, cross-striped cabbageworm, european corn borer, diamondbackmoth, green cloverworm, imported cabbageworm, melonworm, omnivorousleafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybeanlooper, tobacco budworm, tomato fruitworm, tomato hornworm, tomatopinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise,pasture and hay crops such as alfalfa, pasture grasses and silage areoften attacked by such pests as armyworm, beef armyworm, alfalfacaterpillar, European skipper, a variety of loopers and webworms, aswell as yellowstriped armyworms.

Field crops such as canola/rape seed, evening primrose, meadow foam,corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice,safflower, small grains (barley, oats, rye, wheat, etc.), sorghum,soybeans, sunflowers, and tobacco are often targets for infestation byinsects including armyworm, asian and other corn borers, bandedsunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm(including southern and western varieties), cotton leaf perforator,diamondback moth, european corn borer, green cloverworm, headmoth,headworm, imported cabbageworm, loopers (including Anacamptodes spp.),obliquebanded leafroller, omnivorous leaftier, podworm, podworm,saltmarsh caterpillar, southwestern corn borer, soybean looper, spottedcutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbeancaterpillar,

Bedding plants, flowers, ornamentals, vegetables and container stock arefrequently fed upon by a host of insect pests such as armyworm, azaleamoth, beet armyworm, diamondback moth, ello moth (hornworm), Floridafern caterpillar, Io moth, loopers, oleander moth, omnivorousleafroller, omnivorous looper, and tobacco budworm.

Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs andother nursery stock are often susceptible to attack from diverse insectssuch as bagworm, blackheaded budworm, browntail moth, californiaoakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittreeleafroller, greenstriped mapleworm, gypsy moth, jack pine budworm,mimosa webworm, pine butterfly, redhumped caterpillar, saddlebackcaterpillar, saddle prominent caterpillar, spring and fall cankerworm,spruce budworm, tent caterpillar, tortrix, and western tussock moth.Likewise, turf grasses are often attacked by pests such as armyworm, sodwebworm, and tropical sod webworm.

Because crops of commercial interest are often the target of insectattack, environmentally-sensitive methods for controlling or eradicatinginsect infestation are desirable in many instances. This is particularlytrue for farmers, nurserymen, growers, and commercial and residentialareas which seek to control insect populations using eco-friendlycompositions.

The most widely used environmentally-sensitive insecticidal formulationsdeveloped in recent years have been composed of microbial pesticidesderived from the bacterium Bacillus thuringiensis. B. thuringiensis is aGram-positive bacterium that produces crystal proteins or inclusionbodies which are specifically toxic to certain orders and species ofinsects. Many different strains of B. thuringiensis have been shown toproduce insecticidal crystal proteins. Compositions including B.thuringiensis strains which produce insecticidal proteins have beencommercially-available and used as environmentally-acceptableinsecticides because they are quite toxic to the specific target insect,but are harmless to plants and other non-targeted organisms.

1.2.1 δ-Endotoxins

δ-endotoxins are used to control a wide range of leaf-eatingcaterpillars and beetles, as well as mosquitoes. These proteinaceousparasporal crystals, also referred to as insecticidal crystal proteins,crystal proteins, Bt inclusions, crystaline inclusions, inclusionbodies, and Bt toxins, are a large collection of insecticidal proteinsproduced by B. thuringiensis that are toxic upon ingestion by asusceptible insect host. Over the past decade research on the structureand function of B. thuringiensis toxins has covered all of the majortoxin categories, and while these toxins differ in specific structureand function, general similarities in the structure and function areassumed. Based on the accumulated knowledge of B. thuringiensis toxins,a generalized mode of action for B. thuringiensis toxins has beencreated and includes: ingestion by the insect, solubilization in theinsect midgut (a combination stomach and small intestine), resistance todigestive enzymes sometimes with partial digestion actually “activating”the toxin binding to the midgut cells, formation of a pore in the insectcells and the disruption of cellular homeostasis (English and Slatin,1992).

1.2.2 Genes Encoding Crystal Proteins

Many of the δ-endotoxins are related to various degrees by similaritiesin their amino acid sequences. Historically, the proteins and the geneswhich encode them were classified based largely upon their spectrum ofinsecticidal activity. The review by Höfte and Whiteley (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins:cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encodeCryII proteins that are toxic to both lepidopterans and dipterans.cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genesencode dipteran-toxic CryIV proteins, etc. Based on the degree ofsequence similarity, the proteins were further classified intosubfamilies; more highly related proteins within each family wereassigned divisional letters such as CryIA, CryIB, CryIC, etc. Even moreclosely related proteins within each division were given names such asCryIC1, CryIC2, etc.

Recently a new nomenclature was developed which systematicallyclassifies the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities. This classificationscheme, including most of the known toxins but not including allelicvariations in individual polypeptides, is summarized in Table 1.

TABLE 1 KNOWN B. THURINGIENSIS δ-ENDOTOXINS, GENBANK ACCESSION NUMBERS,AND REVISED NOMENCLATURE^(A) New Old GenBank Accession # Cry1Aa1CryIA(a) M11250 Cry1Aa2 CryIA(a) M10917 Cry1Aa3 CryIA(a) D00348 Cry1Aa4CryIA(a) X13535 Cry1Aa5 CryIA(a) D17518 Cry1Aa6 CryIA(a) U43605 Cry1Ab1CryIA(b) M13898 Cry1Ab2 CryIA(b) M12661 Cry1Ab3 CryIA(b) M15271 Cry1Ab4CryIA(b) D00117 Cry1Ab5 CryIA(b) X04698 Cry1Ab6 CryIA(b) M37263 Cry1Ab7CryIA(b) X13233 Cry1Ab8 CryIA(b) M16463 Cry1Ab9 CryIA(b) X54939 Cry1Ab10CryIA(b) Cry1Ac1 CryIA(c) M11068 Cry1Ac2 CryIA(c) M35524 Cry1Ac3CryIA(c) X54159 Cry1Ac4 CryIA(c) M73249 Cry1Ac5 CryIA(c) M73248 Cry1Ac6CryIA(c) U43606 Cry1Ac7 CryIA(c) U87793 Cry1Ac8 CryIA(c) U87397 Cry1Ac9CryIA(c) U89872 Cry1Ac10 CryIA(c) AJ002514 Cry1Ad1 CryIA(d) M73250Cry1Ae1 CryIA(e) M65252 Cry1Ba1 CryIB X06711 Cry1Ba2 X95704 Cry1Bb1 ET5L32020 Cry1Bc1 CryIb(c) Z46442 Cry1Bd1 CryE1 Cry1Ca1 CryIC X07518Cry1Ca2 CryIC X13620 Cry1Ca3 CryIC M73251 Cry1Ca4 CryIC A27642 Cry1Ca5CryIC X96682 Cry1Ca6 CryIC X96683 Cry1Ca7 CryIC X96684 Cry1Cb1 CryIC(b)M97880 Cry1Da1 CryID X54160 Cry1Db1 PrtB Z22511 Cry1Ea1 CryIE X53985Cry1Ea2 CryIE X56144 Cry1Ea3 CryIE M73252 Cry1Ea4 U94323 Cry1Eb1CryIE(b) M73253 Cry1Fa1 CryIF M63897 Cry1Fa2 CryIF M63897 Cry1Fb1 PrtDZ22512 Cry1Ga1 PrtA Z22510 Cry1Ga2 CryIM Y09326 Cry1Gb1 CryH2 Cry1Ha1PrtC Z22513 Cry1Hb1 U35780 Cry1Ia1 CryV X62821 Cry1Ia2 CryV M98544Cry1Ia3 CryV L36338 Cry1Ia4 CryV L49391 Cry1Ia5 CryV Y08920 Cry1Ib1 CryVU07642 Cry1Ja1 ET4 L32019 Cry1Jb1 ET1 U31527 Cry1Ka1 U28801 Cry2Aa1CryIIA M31738 Cry2Aa2 CryIIA M23723 Cry2Aa3 D86084 Cry2Ab1 CryIIB M23724Cry2Ab2 CryIIB X55416 Cry2Ac1 CryIIC X57252 Cry3Aa1 CryIIIA M22472Cry3Aa2 CryIIIA J02978 Cry3Aa3 CryIIIA Y00420 Cry3Aa4 CryIIIA M30503Cry3Aa5 CryIIIA M37207 Cry3Aa6 CryIIIA U10985 Cry3Ba1 CryIIIB X17123Cry3Ba2 CryIIIB A07234 Cry3Bb1 CryIIIB2 M89794 Cry3Bb2 CryIIIC(b) U31633Cry3Ca1 CryIIID X59797 Cry4Aa1 CryIVA Y00423 Cry4Aa2 CryIVA D00248Cry4Ba1 CryIVB X07423 Cry4Ba2 CryIVB X07082 Cry4Ba3 CryIVB M20242Cry4Ba4 CryIVB D00247 Cry5Aa1 CryVA(a) L07025 Cry5Ab1 CryVA(b) L07026Cry5Ba1 PS86Q3 U19725 Cry6Aa1 CryVIA L07022 Cry6Ba1 CryVIB L07024Cry7Aa1 CryIIIC M64478 Cry7Ab1 CryIIICb U04367 Cry8Aa1 CryIIIE U04364Cry8Ba1 CryIIIG U04365 Cry8Ca1 CryIIIF U04366 Cry9Aa1 CryIG X58120Cry9Aa2 CryIG X58534 Cry9Ba1 CryIX X75019 Cry9Ca1 CryIH Z37527 Cry9Da1N141 D85560 Cry10Aa1 CryIVC M12662 Cry11Aa1 CryIVD M31737 Cry11Aa2CryIVD M22860 Cry11Ba1 Jeg80 X86902 Cry12Aa1 CryVB L07027 Cry13Aa1 CryVCL07023 Cry14Aa1 CryVD U13955 Cry15Aa1 34 kDa M76442 Cry16Aa1 cbm71X94146 Cry17Aa1 cbm71 X99478 Cry18Aa1 CryBP1 X99049 Cry19Aa1 Jeg65Y08920 Cry20Aa1 U82518 Cry21Aa1 I32932 Cry22Aa1 I34547 Cyt1Aa1 CytAX03182 Cyt1Aa2 CytA X04338 Cyt1Aa3 CytA Y00135 Cyt1Aa4 CytA M35968Cyt1Ab1 CytM X98793 Cyt1Ba1 U37196 Cyt2Aa1 CytB Z14147 Cyt2Ba1 “CytB”U52043 Cyt2Ba2 “CytB” AF020789 Cyt2Ba3 “CytB” AF022884 Cyt2Ba4 “CytB”AF022885 Cyt2Ba5 “CytB” AF022886 Cyt2Bb1 U82519 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html1.2.3 Bioinsecticide Polypeptide Compositions

The utility of bacterial crystal proteins as insecticides was extendedbeyond lepidopterans and dipteran larvae when the first isolation of acoleopteran-toxic B. thuringiensis strain was reported (Krieg et al.,1983; 1984). This strain (described in U.S. Pat. No. 4,766,203,specifically incorporated herein by reference), designated B.thuringiensis var. tenebrionis, is reported to be toxic to larvae of thecoleopteran insects Agelastica alni (blue alder leaf beetle) andLeptinotarsa decemlineata (Colorado potato beetle).

U.S. Pat. No. 5,024,837 also describes hybrid B. thuringiensis var.kurstaki strains which showed activity against lepidopteran insects.U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybridB. thuringiensis containing a plasmid from B. thuringiensis var.kurstaki encoding a lepidopteran-toxic crystal protein-encoding gene anda plasmid from B. thuringiensis tenebrionis encoding a coleopteran-toxiccrystal protein-encoding gene. The hybrid B. thuringiensis strainproduces crystal proteins characteristic of those made by both B.thuringiensis kurstaki and B. thuringiensis tenebrionis. U.S. Pat. No.4,910,016 (corresponding to EP 0303379) discloses a B. thuringiensisisolate identified as B. thuringiensis MT 104 which has insecticidalactivity against coleopterans and lepidopterans.

1.2.4 Molecular Genetic Techniques Facilitate Protein Engineering

The revolution in molecular genetics over the past decade hasfacilitated a logical and orderly approach to engineering proteins withimproved properties. Site specific and random mutagenesis methods, theadvent of polymerase chain reaction (PCR™) methodologies, and relatedadvances in the field have permitted an extensive collection of toolsfor changing both amino acid sequence, and underlying genetic sequencesfor a variety of proteins of commercial, medical, and agriculturalinterest.

Following the rapid increase in the number and types of crystal proteinswhich have been identified in the past decade, researchers began totheorize about using such techniques to improve the insecticidalactivity of various crystal proteins. In theory, improvements toδ-endotoxins should be possible using the methods available to proteinengineers working in the art, and it was logical to assume that it wouldbe possible to isolate improved variants of the wild-type crystalproteins isolated to date. By strengthening one or more of theaforementioned steps in the mode of action of the toxin, improvedmolecules should provide enhanced activity, and therefore, represent abreakthrough in the field. If specific amino acid residues on theprotein are identified to be responsible for a specific step in the modeof action, then these residues can be targeted for mutagenesis toimprove performance

1.2.5 Structural Analyses of Crystal Proteins

The combination of structural analyses of B. thuringiensis toxinsfollowed by an investigation of the function of such structures, motifs,and the like has taught that specific regions of crystal proteinendotoxins are, in a general way, responsible for particular functions.

Domain 1, for example, from Cry3Bb and Cry1Ac has been found to beresponsible for ion channel activity, the initial step in formation of apore (Walters et al., 1993; Von Tersch et al., 1994). Domains 2 and 3have been found to be responsible for receptor binding and insecticidalspecificity (Aronson et al., 1995; Caramori et al., 1991; Chen et al.1993; de Maagd et al., 1996; Ge et al., 1991; Lee et al., 1992; Lee etal., 1995; Lu et al., 1994; Smedley and Ellar, 1996; Smith and Ellar,1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu and Dean,1996). Regions in domain 2 and 3 can also impact the ion channelactivity of some toxins (Chen et al., 1993, Wolfersberger et al., 1996;Von Tersch et al., 1994).

1.3 Deficiencies in the Prior Art

Unfortunately, while many laboratories have attempted to make mutatedcrystal proteins, few have succeeded in making mutated crystal proteinswith improved lepidopteran toxicity. In almost all of the examples ofgenetically-engineered B. thuringiensis toxins in the literature, thebiological activity of the mutated crystal protein is no better thanthat of the wild-type protein, and in many cases, the activity isdecreased or destroyed altogether (Almond and Dean, 1993; Aronson etal., 1995; Chen et al., 1993, Chen et al., 1995; Ge et al., 1991; Kwaket al., 1995; Lu et al., 1994; Rajamohan et al., 1995; Rajamohan et al.,1996; Smedley and Ellar, 1996; Smith and Ellar, 1994; Wolfersberger etal., 1996; Wu and Aronson, 1992).

For a crystal protein having approximately 650 amino acids in thesequence of its active toxin, and the possibility of 20 different aminoacids at each position in this sequence, the likelihood of arbitrarilycreating a successful new structure is remote, even if a generalfunction to a stretch of 250-300 amino acids can be assigned. Indeed,the above prior art with respect to crystal protein gene mutagenesis hasbeen concerned primarily with studying the structure and function of thecrystal proteins, using mutagenesis to perturb some step in the mode ofaction, rather than with engineering improved toxins.

Collectively, the limited successes in the art to develop synthetictoxins with improved insecticidal activity have stifled progress in thisarea and confounded the search for improved endotoxins or crystalproteins. Rather than following simple and predictable rules, thesuccessful engineering of an improved crystal protein may involvedifferent strategies, depending on the crystal protein being improvedand the insect pests being targeted. Thus, the process is highlyempirical.

Accordingly, traditional recombinant DNA technology is clearly notroutine experimentation for providing improved insecticidal crystalproteins. What are lacking in the prior art are rational methods forproducing genetically-engineered B. thuringiensis crystal proteins thathave improved insecticidal activity and, in particular, improvedtoxicity towards a wide range of lepidopteran insect pests.

2.0 SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacksinherent in the prior art by providing genetically-engineered modifiedB. thuringiensis δ-endotoxins (Cry*), and in particular modified Cry3δ-endotoxins (designated Cry3* endotoxins). Also provided are nucleicacid sequences comprising one or more genes which encode such modifiedproteins. Particularly preferred genes include cry3* genes such ascry3A*, cry3B*, and cry3C* genes, particularly cry3B* genes, and moreparticularly, cry3Bb* genes, that encode modified crystal proteinshaving improved insecticidal activity against target pests.

Also disclosed are novel methods for constructing synthetic Cry3*proteins, synthetically-modified nucleic acid sequences encoding suchproteins, and compositions arising therefrom. Also provided aresynthetic cry3* expression vectors and various methods of using theimproved genes and vectors. In a preferred embodiment, the inventiondiscloses and claims Cry3B* proteins and cry3B* genes which encodeimproved insecticidal polypeptides.

In preferred embodiments, channel-forming toxin design methods aredisclosed which have been used to produce a specific set of designedCry3Bb* toxins with improved biological activity. These improved Cry3Bb*proteins are listed in Table 2 along with their respective amino acidchanges from wild-type (WT) Cry3Bb, the nucleotide changes present inthe altered cry3Bb* gene encoding the protein, the fold increase inbioactivity over WT Cry3Bb, the structural site of the alteration, andthe design method(s) used to create the new toxins.

Accordingly, the present invention provides in an overall and generalsense, mutagenized Cry3 protein-encoding genes and methods of making andusing such genes. As used herein the term “mutagenized cry3 gene(s)”means one or more cry3 genes that have been mutagenized or altered tocontain one or more nucleotide sequences which are not present in thewild type sequences, and which encode mutant Cry3 crystal proteins(Cry3*) showing improved insecticidal activity. Such mutagenized cry3genes have been referred to in the Specification as cry3* genes.Exemplary cry3* genes include cry3A*, cry3B*, and cry3C* genes.

Exemplary mutagenized Cry3 protein-encoding genes include cry3B genes.As used herein the term “mutagenized cry3B gene(s)” means one or moregenes that have been mutagenized or altered to contain one or morenucleotide sequences which are not present in the wild type sequences,and which encode mutant Cry3B crystal proteins (Cry3B*) showing improvedinsecticidal activity. Such genes have been designated cry3B* genes.Exemplary cry3B* genes include cry3Ba* and cry3Bb* genes, which encodeCry3Ba* and Cry3Bb* proteins, respectively.

Likewise, the present invention provides mutagenized Cry3Aprotein-encoding genes and methods of making and using such genes. Asused herein the term “mutagenized cry3A gene(s)” means one or more genesthat have been mutagenized or altered to contain one or more nucleotidesequences which are not present in the wild type sequences, and whichencode mutant Cry3A crystal proteins (Cry3A*) showing improvedinsecticidal activity. Such mutagenized genes have been designated ascry3A* genes.

In similar fashion, the present invention provides mutagenized Cry3Cprotein-encoding genes and methods of making and using such genes. Asused herein the term “mutagenized cry3C gene(s)” means one or more genesthat have been mutagenized or altered to contain one or more nucleotidesequences which are not present in the wild type sequences, and whichencode mutant Cry3C crystal proteins (Cry3C*) showing improvedinsecticidal activity. Such mutagenized genes have been designated ascry3C* genes.

Preferably the novel sequences comprise nucleic acid sequences in whichat least one, and preferably, more than one, and most preferably, asignificant number, of wild-type cry3 nucleotides have been replacedwith one or more nucleotides, or where one or more nucleotides have beenadded to or deleted from the native nucleotide sequence for the purposeof altering, adding, or deleting the corresponding amino acids encodedby the nucleic acid sequence so mutagenized. The desired result,therefore, is alteration of the amino acid sequence of the encodedcrystal protein to provide toxins having improved or altered activityand/or specificity compared to that of the unmodified crystal protein.

Examples of preferred Cry2Bb*-encoding genes include cry3Bb.60,cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,cry3Bb.11226. cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230,cry3Bb.11231, cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235,cry3Bb.11236, cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241,cry3Bb.11242, cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081,cry3Bb. 11082, cry3Bb.11083, cry3Bb.11084, cry3Bb.11095, andcry3Bb.11098.

TABLE 2 CRY3BB* PROTEINS EXHIBITING IMPROVED ACTIVITY AGAINST SCRWLARVAE Cry3Bb* cry3Bb* Structural Fold Design Protein Plasmid cry3Bb*Nucleotide Sequence Cry3Bb* Amino Site Increase Over Method DesignationDesignation Changes Acid Changes of Changes WT Activity Used Cry3Bb.60 —— Δ1–159 Δα1–α3 3.6× 1, 6, 8 Cry3Bb.11221 pEG1707 A460T, C461T, A462T,C464A, T154F, P155H, 1α3, 4 6.4× 1, 8 T465C, T466C, T467A, A468T, L156H,L158R A469T, G470C, T472C, T473G, G474T, A477T, A478T, G479CCry3Bb.11222 pEG1708 T687C, T688C, A689T, C691A, Y230L, H231S α6 4.0× 3,7 A692G Cry3Bb.11223 pEG1709 T667C, T687C, T688A, A689G, S223P, Y230S α62.8× 3 C691A, A692G Cry3Bb.11224 pEG1710 T687C, A692G H231R α6 5.0× 7, 8Cry3Bb.11225 pEG1711 T687C, C691A H231N, T241S α6 3.6× 7 Cry3Bb.11226pEG1712 T687C, C691A, A692C, T693C H231T α6 3.0× 7, 8 Cry3Bb.11227pEG1713 C868A, G869A, G870T R290N 1α7, β1 1.9× 2, 3, 4, 6 Cry3Bb.11228pEGI714 C932T, A938C, T942G, G949A, S311L, N313T, 1β1, α8 4.1× 2, 4T954C E317K Cry3Bb.11229 pEG1715 T931A, A933C, T942A, T945A, S311T,E317K, 1β1, α8 2.5× 2, 4 G949A, A953G, T954C Y318C Cry3Bb.11230 pEG1716T931G, A933C, C934G, T945G, S311A, L312V, 1β1, α8 4.7× 2, 4 8 C946T,A947G, G951A, T954C Q316W Cry3Bb.11231 pEG1717 T687C, A692G, C932T,A938C, H231R, S311L, α6; 1β1, α8 7.9× 2, 4, 7, 8, T942G, G949A, T954CN313T, E317K 10 Cry3Bb.11232 pEG1718 T931A, A933G, T935C, T936A, S311T,L312P, 1β1, α8 5.1× 4 A938C, T939C, T942C, T945A, N313T, E317N G951T,T954C Cry3Bb.11233 pEG1719 T931G, A933C, T936G, T942C, S311A, Q316D 1β1,α8 2.2× 2, 4 C943T, T945A, C946G, G948C, T954C Cry3Bb.11234 pEG1720T861C, T866C, C868A, T871C, 1289T, L291R, 1α7, β1 4.1× 4 T872G, A875T,T877A, C878G, Y292F, S293R A882G Cry3Bb.11235 pEG1721 T687C, A692G,C932T H231R, S311L α6; 1β1, α8 3.2× 2, 4, 7, 8, 10 Cry3Bb.11236 pEG1722T931A, C932T, A933C, T936C, S311I 1β1, α8 3.1× 2, 4 T942G, T945A, T954CCry3Bb.11237 pEG1723 T931A, C932T, A933C, T936C, S311I, N313H 1β1, α85.4× 2, 4 A937G, A938T, C941A, T942C, T945A, C946A, A947T, A950T, T954CCry3Bb.11238 pEG1724 A933C, T936C, A937G, A938T, N313V, T314N, 1β1, α82.6× 2, 4 C941A, T942C, T945A, C946A, Q316M, E317V A947T, A950T, T954CCry3Bb.11239 pEG1725 A933T, A938G, T939G, T942A, N313R, L315P, 1β1, α82.8× 2, 4 T944C, T945A, A947T, G948T, Q316L, E317A A950C, T954CCry3Bb.11241 pEG1726 A860T, T861C, G862A, C868T, Y287F, D288N, 1α7, β12.6× 2, 3, 4, 6 G869T, T871C, A873T, T877A, R290L C878G, A879TCry3Bb.11242 pEG1727 C868G, G869T R290V 1α7, β1 2.5× 2, 3, 4, 6, 8Cry3Bb.11032 pEG1041 A494G D165G α4 3.1× 2, 4, 8 Cry3Bb.11035 pEG1046G479A, A481C, A482C, S160N, K161P, α4 2.7× 8 A484C, G485A, A486C, A494GR162H, D165G Cry3Bb.11036 pEG1047 A865G, T877C I289V, S293P 1α7, β1 4.3×4 Cry3Bb.11046 pEG1052 G479A, A481C, A482C, S160N, K161P, α4; 1α7, β12.6× 2, 4, 8, 10 A484C, G485A, A486C, R162H, D165G, A494G, A865G, T877CI289V, S293P Cry3Bb.11048 pEG1054 T309A, Δ310, Δ311, Δ312 D103E, ΔA1041α2a, 2b 4.3× 8 Cry3Bb.11051 pEG1057 A565G, A566G K189G 1α4, 5 3.0× 2,3, 4 Cry3Bb.11057 pEG1062 T309A, Δ310, Δ311, Δ312, D103E, ΔA104, 1α2a,2b; α4 3.4× 2, 4, 8, 10 G479A, A481C, A482C, S160N, K161P, A484C, G485A,A486C, A494G R162H, D165G Cry3Bb.11058 pEG1063 T309A, Δ310, Δ311, Δ312,D103E, ΔA104, 1α2a, 2b; 3.5× 1, 8, 10 A460T, C461T, A462T, C464A, T154F,P155H, 1α3, 4 T465C, T466C, T467A, A468T, L156H, L158R A469T, G470C,T472C, T473G, G474T, A477T, A478T, G479C Cry3Bb.11081 pEG1084 A494G,T931A, A933C, D165G, S311T, α4; 1β1, α8 6.1× 2, 4, 8, 10 T942A, T945A,G949A, T954C E317K Cry3Bb.11082 pEG1085 A494G, A865G, T877C, T914C,D165G, I289V, α4; 1α7, β1; 4.9× 2, 4, 5, 8, β1; T931G, A933C, C934G,T945G, S293P, F305S, 1β1, α8; β2; 9, 10 C946T, A947G, G951A, T954C,S311A, L312V, β3b A1043G, T1094C Q316W, Q348R, V365A Cry3Bb.11083pEG1086 A865G, T877C, A1043G I289V, S293P, 1α7, β1; β2 7.4× 4, 5, 9, 10Q348R Cry3Bb.11084 pEG1087 A494G, C932T D165G, S311L α4; 1β1, α8 7.2× 2,4, 8, 10 Cry3Bb.11095 pEG1095 A1043G Q348R β2 4.6× 5, 9 Cry3Bb.11098pEG1098 A494G, T687C, A692G, C932T, D165G, H231R, α4; α6, 1β1, 7.9× 2,4, 7, 8 A938C, T942G, G949A, T954C S311L, N313T, α8 E317K

In a variety of illustrative embodiments, the inventors have shownremarkable success in generating toxins with improved insecticidalactivity using these methods. In particular, the inventors haveidentified unique methods of analyzing and designing toxins havingimproved or enhanced insecticidal properties both in vitro and in vivo.

In addition to modifications of Cry3Bb peptides, those having benefit ofthe present teaching are now also able to make mutations in a variety ofchannel-forming toxins, and particularly in crystal proteins which arerelated to Cry3Bb either functionally or structurally. In fact, theinventors contemplate that any B. thuringiensis crystal protein orpeptide can be analyzed using the methods disclosed herein and may bealtered using the methods disclosed herein to produce crystal proteinshaving improved insecticidal specificity or activity. Alternatively, theinventors contemplate that those of skill in the art having the benefitof the teachings disclosed herein will be able to prepare not onlymutated Cry3 toxins with improved activity, but also other crystalproteins including all of those proteins identified in Table 1, herein.In particular, the inventors contemplate the creation of Cry3* variantsusing one or more of the methods disclosed herein to produce toxins withimproved activity. For example, the inventors note Cry3A, Cry3B, andCry3C crystal proteins (which are known in the art) may be modifiedusing one or more of the design strategies employed herein, to preparesynthetically-modifiedcrystal proteins with improved properties.Likewise, one of skill in the art will even be able to utilize theteachings of the present disclosure to modify other channel formingtoxins, including channel forming toxins other than B. thuringiensiscrystal proteins, and even to modify proteins and channel toxins not yetdescribed or characterized.

Because the structures for insecticidal crystal proteins show aremarkable conservation of protein tertiary structure (Grochulski etal., 1995), and because many crystal proteins show significant aminoacid sequence identity to the Cry3Bb amino acid sequence within domain1, including proteins of the Cry1, Cry2, Cry3, Cry4, Cry5, Cry7, Cry8,Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, and Cry16 classes (Table 1),now in light of the inventors' surprising discovery, for the first time,those of skill in the art having benefit of the teachings disclosedherein will be able to broadly apply the methods of the invention tomodifying a host of crystal proteins with improved activity or alteredspecificity. Such methods will not only be limited to the insecticidalcrystal proteins disclosed in Table 1, but may also been applied to anyother related crystal protein, including those yet to be identified.

In particular, the high degree of homology between Cry3A, Cry3B, andCry3C proteins is evident in the alignment of the primary amino acidsequence of the three proteins (FIG. 17A, FIG. 17B, and FIG. 17C).

As such, the disclosed methods may be now applied to preparation ofmodified crystal proteins having one or more alterations introducedusing one or more of the mutational design methods as disclosed herein.The inventors further contemplate that regions may be identified in oneor more domains of a crystal protein, or other channel forming toxinwhich may be similarly modified through site-specific or randommutagenesis to generate toxins having improved activity, oralternatively, altered specificity.

In certain applications, the creation of altered toxins having increasedactivity against one or more insects is desired. Alternatively, it maybe desirable to utilize the methods described herein for creating andidentifying altered insecticidal crystal proteins which are activeagainst a wider spectrum of susceptible insects. The inventors furthercontemplate that the creation of chimeric insecticidal crystal proteinscomprising one or more of these mutations may be desirable for preparing“super” toxins which have the combined advantages of increasedinsecticidal activity and concomitant broad spectrum activity.

In light of the present disclosure, the mutagenesis of one or morecodons within the sequence of a toxin may result in the generation of ahost of related insecticidal proteins having improved activity. Whileexemplary mutations have been described for each of the designstrategies employed in the present invention, the inventors contemplatethat mutations may also be made in insecticidal crystal proteins,including the loop regions, helices regions, active sites of the toxins,regions involved in protein oligomerization, and the like, which willgive rise to functional bioinsecticidal crystal proteins. All suchmutations are considered to fall within the scope of this disclosure.

In one illustrative embodiment, mutagenized cry3Bb* genes are obtainedwhich encode Cry3Bb* variants that are generally based upon thewild-type Cry3Bb sequence, but that have one or more changesincorporated into the amino acid sequence of the protein using one ormore of the design strategies described and claimed herein.

In these and other embodiments, the mutated genes encoding the crystalproteins may be modified so as to change about one, two, three, four, orfive or so amino acids in the primary sequence of the encodedpolypeptide. Alternatively even more changes from the native sequencemay be introduced, such that the encoded protein may have at least about1% or 2%, or alternatively about 3% or about 4%, or even about 5% toabout 10%, or about 10% to about 15%, or even about 15% to about 20% ormore of the codons either altered, deleted, or otherwise modified. Incertain situations, it may even be desirable to alter substantially moreof the primary amino acid sequence to obtain the desired modifiedprotein. In such cases the inventors contemplate that from about 25%, toabout 50%, or even from about 50% to about 75%, or more of the native(or wild-type) codons either altered, deleted, or otherwise modified.Alternatively, mutations in the amino acid sequences or underlying DNAgene sequences which result in the insertion or deletion of one or moreamino acids within one or more regions of the crystal protein orpeptide.

To effect such changes in the primary sequence of the encodedpolypeptides, it may be desirable to mutate or delete one or morenucleotides from the nucleic acid sequences of the genes encoding suchpolypeptides, or alternatively, under certain circumstances to add oneor more nucleotides into the primary nucleic acid sequence at one ormore sites in the sequence. Frequently, several nucleotide residues maybe altered to produce the desired polypeptide. As such, the inventorscontemplate that in certain embodiments it may be desirable to alteronly one, two, three, four, or five or so nucleotides in the primarysequence. In other embodiments, which more changes are desired, themutagenesis may involve changing, deleting, or inserting 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or even 20 or so nucleotide residuesin the gene sequence. In still other embodiments, one may desire tomutate, delete, or insert 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, or even 90-100, 150, 200, 250, 300,350, 400, 450, or more nucleotides in the sequence of the gene in orderto prepare a cry3* gene which produces a Cry3* polypeptide having thedesired characteristics. In fact, any number of mutations, deletions,and/or insertions may be made in the primary sequence of the gene, solong as the encoded protein has the improved insecticidal activity orspecificity characteristics described herein.

Changing a large number of the codons in the nucleotide sequence of anendotoxin-encoding gene may be particularly desirable and oftennecessary to achieve the desired results, particularly in the situationof “plantizing” a DNA sequence in order to express a DNA of non-plantorigin in a transformed plant cell. Such methods are routine to those ofskill in the plant genetics arts, and frequently many residues of aprimary gene sequence will be altered to facilitate expression of thegene in the plant cell. Preferably, the changes in the gene sequenceintroduce no changes in the amino acid sequence, or introduce onlyconservative replacements in the amino acid sequence such that thepolypeptide produced in the plant cell from the “plantized” nucleotidesequence is still fully functional, and has the desired qualities whenexpressed in the plant cell.

Genes and encoded proteins mutated in the manner of the invention mayalso be operatively linked to other protein-encoding nucleic acidsequences, or expressed as fusion proteins. Both N-terminal andC-terminal fusion proteins are contemplated. Virtually any protein- orpeptide-encoding DNA sequence, or combinations thereof, may be fused toa mutated cry3* sequence in order to encode a fusion protein. Thisincludes DNA sequences that encode targeting peptides, proteins forrecombinant expression, proteins to which one or more targeting peptidesis attached, protein subunits, domains from one or more crystalproteins, and the like. Such modifications to primary nucleotidesequences to enhance, target, or optimize expression of the genesequence in a particular host cell, tissue, or cellular localization,are well-known to those of skill in the art of protein engineering andmolecular biology, and it will be readily apparent to such artisans,having benefit of the teachings of this specification, how to facilitatesuch changes in the nucleotide sequence to produce the polypeptides andpolynucleotides disclosed herein.

In one aspect, the invention discloses and claims host cells comprisingone or more of the modified crystal proteins disclosed herein, and inparticular, cells of B. thuringiensis strains EG11221, EG11222, EG11223,EG11224, EG11225, EG11226, EG11227, EG11228, EG11229, EG11230, EG11231,EG11232, EG11233, EG11234, EG11235, EG11236, EG11237, EG11238, EG11239,EG11241, EG11242, EG11032, EG11035, EG11036, EG11046, EG11048, EG11051,EG11057, EG11058, EG11081, EG11082, EG11083, EG11084, EG11095, andEG11098 which comprise recombinant DNA segments encodingsynthetically-modified Cry3 Bb* crystal proteins which demonstratesimproved insecticidal activity.

Likewise, the invention also discloses and claims cell cultures of B.thuringiensis EG11221, EG11222, EG11223, EG11224, EG11225, EG11226,EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233, EG11234,EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242, EG11032,EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058, EG11081,EG11082, EG11083, EG11084, and EG11095, and 11098.

Such cell cultures may be biologically-pure cultures consisting of asingle strain, or alternatively may be cell co-cultures consisting ofone or more strains. Such cell cultures may be cultivated underconditions in which one or more additional B. thuringiensis or otherbacterial strains are simultaneously co-cultured with one or more of thedisclosed cultures, or alternatively, one or more of the cell culturesof the present invention may be combined with one or more additional B.thuringiensis or other bacterial strains following the independentculture of each. Such procedures may be useful when suspensions of cellscontaining two or more different crystal proteins are desired.

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35U.S.C. §122. The deposits are available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the finishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Cultures shown in Table 3 were deposited in the permanent collection ofthe Agricultural Research Service Culture Collection, Northern RegionalResearch Laboratory (NRRL) under the terms of the Budapest Treaty.

TABLE 3 STRAINS OF THE PRESENT INVENTION DEPOSITED UNDER THE TERMS OFTHE BUDAPEST TREATY Accession Number Strain Deposit Date Protein (NRRLNumber) EG11032 May 27, 1997 Cry3Bb.11032 B-21744 EG11035 May 27, 1997Cry3Bb.11035 B-21745 EG11036 May 27, 1997 Cry3Bb.11036 B-21746 EG11037May 27, 1997 Cry3Bb.11037 B-21747 EG11046 May 27, 1997 Cry3Bb.11046B-21748 EG11048 May 27, 1997 Cry3Bb.11048 B-21749 EG11051 May 27, 1997Cry3Bb.11051 B-21750 EG11057 May 27, 1997 Cry3Bb.11057 B-21751 EG11058May 27, 1997 Cry3Bb.11058 B-21752 EG11081 May 27, 1997 Cry3Bb.11081B-21753 EG11082 May 27, 1997 Cry3Bb.11082 B-21754 EG11083 May 27, 1997Cry3Bb.11083 B-21755 EG11084 May 27, 1997 Cry3Bb.11084 B-21756 EG11095May 27, 1997 Cry3Bb.11095 B-21757 EG11204 May 27, 1997 Cry3Bb.11204B-21758 EG11221 May 27, 1997 Cry3Bb.11221 B-21759 EG11222 May 27, 1997Cry3Bb.11222 B-21760 EG11223 May 27, 1997 Cry3Bb.11223 B-21761 EG11224May 27, 1997 Cry3Bb.11224 B-21762 EG11225 May 27, 1997 Cry3Bb.11225B-21763 EG11226 May 27, 1997 Cry3Bb.11226 B-21764 EG11227 May 27, 1997Cry3Bb.11227 B-12765 EG11228 May 27, 1997 Cry3Bb.11228 B-12766 EG11229May 27, 1997 Cry3Bb.11229 B-21767 EG11230 May 27, 1997 Cry3Bb.11230B-21768 EG11231 May 27, 1997 Cry3Bb.11231 B-21769 EG11232 May 27, 1997Cry3Bb.11232 B-12770 EG11233 May 27, 1997 Cry3Bb.11233 B-21771 EG11234May 27, 1997 Cry3Bb.11234 B-21772 EG11235 May 27, 1997 Cry3Bb.11235B-21773 EG11236 May 27, 1997 Cry3Bb.11236 B-21774 EG11237 May 27, 1997Cry3Bb.11237 B-21775 EG11238 May 27, 1997 Cry3Bb.11238 B-21776 EG11239May 27, 1997 Cry3Bb.11239 B-21777 EG11241 May 27, 1997 Cry3Bb.11241B-21778 EG11242 May 27, 1997 Cry3Bb.11242 B-21779

Also disclosed are methods of controlling or eradicating an insectpopulation from an environment. Such methods generally comprisecontacting the insect population to be controlled or eradicated with aninsecticidally-effective amount of a Cry3* crystal protein composition.Preferred Cry3* compositions include Cry3A*, Cry3B*, and Cry3C*polypeptide compositions, with Cry3B* compositions being particularlypreferred. Examples of such polypeptides include proteins selected fromthe group consisting of Cry3Bb-60, Cry3Bb.11221, Cry3Bb.11222,Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227,Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232,Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237,Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032,Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051,Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083,Cry3Bb.11084, Cry3Bb.11095, and Cry3Bb.11098.

In preferred embodiments, these Cry3Bb* crystal protein compositionscomprise the amino acid sequence of any of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14. SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102 or SEQID NO:108.

2.1 Methods for Producing Modified Cry* Proteins

The modified Cry* polypeptides of the present invention are preparableby a process which generally involves the steps of obtaining a nucleicacid sequence encoding a Cry* polypeptide; analyzing the structure ofthe polypeptide to identify particular “target” sites for mutagenesis ofthe underlying gene sequence; introducing one or more mutations into thenucleic acid sequence to produce a change in one or more amino acidresidues in the encoded polypeptide sequence; and expressing in atransformed host cell the mutagenized nucleic acid sequence underconditions effective to obtain the modified Cry* protein encoded by thecry* gene.

Means for obtaining the crystal structures of the polypeptides of theinvention are well-known. Exemplary high resolution crystal structuresolution sets are given in Section 9.0 of the disclosure, and includethe crystal structure of both the Cry3A and Cry3B polypeptides disclosedherein. The information provided in Section 9.0 permits the analysesdisclosed in each of the methods herein which rely on the 3D crystalstructure information for targeting mutagenesis of the polypeptides toparticular regions of the primary amino acid sequences of theδ-endotoxins to obtain mutants with increased insecticidal activity orenhanced insecticidal specificity.

A first method for producing a modified B. thuringiensis Cry3Bbδ-endotoxin having improved insecticidal activity or specificitydisclosed herein generally involves obtaining a high-resolution 3Dcrystal structure of the endotoxin, locating in the crystal structureone or more regions of bound water wherein the bound water forms acontiguous hydrated surfaces separated by no more than about 16 Å;increasing the number of water molecules in this surface by increasingthe hydrophobicity of one or more amino acids of the protein in theregion; and obtaining the modified δ-endotoxin so produced. Exemplaryδ-endotoxins include Cry3Bb.11032, Cry3Bb.11227, Cry3Bb.11241,Cry3Bb.11051, Cry3Bb.11242, and Cry3Bb.11098.

A second method for producing a modified B. thuringiensis Cry3Bbδ-endotoxin having improved insecticidal activity comprises identifyinga loop region in a δ-endotoxin; modifying one or more amino acids in theloop to increase the hydrophobicity of the amino acids; and obtainingthe modified δ-endotoxin so produced. Preferred δ-endotoxinproduced bythis method include Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11228,Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11233, Cry3Bb.11236,Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.

A method for increasing the mobility of channel forming helices of a B.thuringiensis Cry3B δ-endotoxin is also provided by the presentinvention. The method generally comprises disrupting one or morehydrogen bonds formed between a first amino acid of one or more of thechannel forming helices and a second amino acid of the δ-endotoxin. Thehydrogen bonds may be formed inter- or intramolecularly, and thedisrupting may consist of replacing a first or second amino acid with athird amino acid whose spatial distance is greater than about 3 Å, orwhose spatial orientation bond angle is not equal to 180±60 degreesrelative to the hydrogen bonding site of the first or second amino acid.δ-endotoxins produced by this method and disclosed herein includeCry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226,Cry3Bb.11227, Cry3Bb.11231, Cry3Bb.11241, and Cry3Bb.11242, andCry3Bb.11098.

Also disclosed is a method of increasing the flexibility of a loopregion in a channel forming domain of a B. thuringiensis Cry3Bbδ-endotoxin. This method comprises obtaining a crystal structure of aCry3Bb δ-endotoxin having one or more loop regions; identifying theamino acids comprising the loop region; and altering one or more of theamino acids to reduce steric hindrance in the loop region, wherein thealtering increases flexibility of the loop region in the δ-endotoxin.Examples of δ-endotoxins produced using this method includeCry3Bb.11032, Cry3Bb.11051, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237,Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241,Cry3Bb.11242, Cry3Bb.11036, and Cry3Bb.11098.

Another aspect of the invention is a method for increasing the activityof a δ-endotoxin, comprising reducing or eliminating binding of theδ-endotoxin to a carbohydrate in a target insect gut. The eliminating orreducing may be accomplished by removal of one or more α helices ofdomain 1 of the δ-endotoxin, for example, by removal of α helices α1,α2a/b, and α3. An exemplary δ-endotoxin produced using the method isCry3Bb.60.

Alternatively, the reducing or eliminating may be accomplished byreplacing one or more amino acids within loop β1,α8, with one or moreamino acids having increased hydrophobicity. Such a method gives rise toδ-endotoxins such as Cry3Bb.11228, Cry3Bb.11230, Cry3B.11231,Cry3Bb.11237, and Cry3Bb.11098, which are described in detail, herein.

Alternatively, the reducing or eliminating is accomplished by replacingone or more specific amino acids, with any other amino acid. Suchreplacements are described in Table 2, and in the examples herein. Oneexample is the δ-endotoxin designated herein as Cry3Bb.11221.

A method of identifying a region of a Cry3Bb δ-endotoxin for targetedmutagenesis comprising: obtaining a crystal structure of theδ-endotoxin; identifying from the crystal structure one or moresurface-exposed amino acids in the protein; randomly substituting one ormore of the surface-exposed amino acids to obtain a plurality of mutatedpolypeptides, wherein at least 50% of the mutated polypeptides havediminished insecticidal activity; and identifying from the plurality ofmutated polypeptides one or more regions of the Cry3Bb δ-endotoxin fortargeted mutagenesis. The method may further comprise determining theamino acid sequences of a plurality of mutated polypeptides havingdiminished activity, and identifying one or more amino acid residuesrequired for insecticidal activity.

In another embodiment, the invention provides a process for producing aCry3Bb δ-endotoxin having improved insecticidal activity. The processgenerally involves the steps of obtaining a high-resolution crystalstructure of the protein; determining the electrostatic surfacedistribution of the protein; identifying one or more regions of highelectrostatic diversity; modifying the electrostatic diversity of theregion by altering one or more amino acids in the region; and obtaininga Cry3Bb δ-endotoxin which has improved insecticidal activity. In oneembodiment, the electrostatic diversity may be decreased relative to theelectrostatic diversity of a native Cry3Bb δ-endotoxin. Exemplaryδ-endotoxins with decreased electrostatic diversity includeCry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242. Alternatively, theelectrostatic diversity may be increased relative to the electrostaticdiversity of a native Cry3Bb δ-endotoxin. An exemplary δ-endotoxin withincreased electrostatic diversity is Cry3Bb.11234.

Furthermore, the invention also provides a method of producing a Cry3Bbδ-endotoxin having improved insecticidal activity which involvesobtaining a high-resolution crystal structure; identifying the presenceof one or more metal binding sites in the protein; altering one or moreamino acids in the binding site; and obtaining an altered protein,wherein the protein has improved insecticidal activity. The altering mayinvolve the elimination of one or more metal binding sites. Exemplaryδ-endotoxin include Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, andCry3Bb.11226.

A further aspect of the invention involves a method of identifying a B.thuringiensis Cry3Bb δ-endotoxin having improved channel activity. Thismethod in an overall sense involves obtaining a Cry3Bb δ-endotoxinsuspected of having improved channel activity; and determining one ormore of the following characteristics in the δ-endotoxin, and comparingsuch characteristics to those obtained for the wild-type unmodifiedδ-endotoxin; (1) the rate of channel formation, (2) the rate of growthof channel conductance or (3) the duration of open channel state. Fromthis comparison, one may then select a δ-endotoxin which has anincreased rate of channel formation compared to the wildtypeδ-endotoxin. Examples of Cry3Bb δ-endotoxins prepared by this methodinclude Cry3Bb.60, Cry3Bb.11035, Cry3Bb.11048, Cry3Bb.11032,Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11226, Cry3Bb.11221, Cry3Bb.11242,Cry3Bb.11230, and Cry3Bb.11098.

Also provided is a method for producing a modified Cry3Bb δ-endotoxin,having improved insecticidal activity which involves altering one ormore non-surface amino acids located at or near the point of greatestconvergence of two or more loop regions of the Cry3Bb δ-endotoxin, suchthat the altering decreases the mobility of one or more of the loopregions. The mobility may conveniently be determined by comparing thethermal denaturation of the modified protein to a wild-type Cry3Bbδ-endotoxin. An exemplary crystal protein produced by this method isCry3Bb.11095.

A further aspect of the invention involves a method for preparing amodified Cry3Bb δ-endotoxin, having improved insecticidal activitycomprising modifying one or more amino acids in the loop to increase thehydrophobicity of said amino acids; and altering one or more of saidamino acids to reduce steric hindrance in the loop region, wherein thealtering increases flexibility of the loop region in the endotoxin.Exemplary Cry3Bb δ-endotoxins produced is selected from the groupconsisting of Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082,Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11231, Cry3Bb.11235, andCry3Bb.11098.

The invention also provides a method of improving the insecticidalactivity of a B. thuringiensis Cry3Bb δ-endotoxin, which generallycomprises inserting one or more protease sensitive sites into one ormore loop regions of domain 1 of the δ-endotoxin. Preferably, the loopregion is α3,4, and an exemplary δ-endotoxin so produced isCry3Bb.11221.

2.2 Polypeptide Compositions

The crystal proteins so produced by each of the methods described hereinalso represent important aspects of the invention. Such crystal proteinspreferably include a protein or peptide selected from the groupconsisting of Cry3Bb-60, Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223,Cry3Bb.11224, Cry3Bb.11225, Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228,Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11234, Cry3Bb.11235, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238,Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035,Cry3Bb.11036, Cry3Bb.11046, Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057,Cry3Bb.11058, Cry3Bb.11081, Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084,Cry3Bb.11095, and Cry3Bb.11098.

In preferred embodiments, the protein comprises a contiguous amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14. SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, and SEQID NO:108.

Highly preferred are those crystal proteins which are encoded by thenucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101; or SEQ ID NO:107, or anucleic acid sequence which hybridizes to the nucleic acid sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO: 11, SEQ ID NO: 13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, SEQ ID NO:101, or SEQ ID NO:107 under conditions of moderatestringency.

Amino acid, peptide and protein sequences within the scope of thepresent invention include, and are not limited to the sequences setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70. SEQ ID NO:100, SEQ ID NO:102, and SEQ ID NO:108, and alterationsin the amino acid sequences including alterations, deletions, mutations,and homologs.

Compositions which comprise from about 0.5% to about 99% by weight ofthe crystal protein, or more preferably from about 5% to about 75%, orfrom about 25% to about 50% by weight of the crystal protein areprovided herein. Such compositions may readily be prepared usingtechniques of protein production and purification well-known to those ofskill, and the methods disclosed herein. Such a process for preparing aCry3Bb* crystal protein generally involves the steps of culturing a hostcell which expresses the Cry3Bb* protein (such as a B. thuringiensisEG11221, EG11222, EG11223, EG11224, EG11225, EG11226, EG11227, EG11228,EG11229, EG11230, EG11231, EG11232, EG11233, EG11234, EG11235, EG11236,EG11237, EG11238, EG11239, EG11241, EG11242, EG11032, EG11035, EG11036,EG11046, EG11048, EG11051, EG11057, EG11058, EG11081, EG11082, EG11083,EG11084, EG11095, or EG11098 cell) under conditions effective to producethe crystal protein, and then obtaining the crystal protein so produced.

The protein may be present within intact cells, and as such, nosubsequent protein isolation or purification steps may be required.Alternatively, the cells may be broken, sonicated, lysed, disrupted, orplasmolyzed to free the crystal protein(s) from the remaining celldebris. In such cases, one may desire to isolate, concentrate, orfurther purify the resulting crystals containing the proteins prior touse, such as, for example, in the formulation of insecticidalcompositions. The composition may ultimately be purified to consistalmost entirely of the pure protein, or alternatively, be purified orisolated to a degree such that the composition comprises the crystalprotein(s) in an amount of from between about 0.5% and about 99% byweight, or in an amount of from between about 5% and about 95% byweight, or in an amount of from between about 15% and about 85% byweight, or in an amount of from between about 25% and about 75% byweight, or in an amount of from between about 40% and about 60% byweight etc.

2.3 Recombinant Vectors Expressing Cry3* Genes

One important embodiment of the invention is a recombinant vector whichcomprises a nucleic acid segment encoding one or more of the novel B.thuringiensis crystal proteins disclosed herein. Such a vector may betransferred to and replicated in a prokaryotic or eukaryotic host, withbacterial cells being particularly preferred as prokaryotic hosts, andplant cells being particularly preferred as eukaryotic hosts.

In preferred embodiments, the recombinant vector comprises a nucleicacid segment encoding the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108. Highly preferred nucleic acid segments arethose which have the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.

Another important embodiment of the invention is a transformed host cellwhich expresses one or more of these recombinant vectors. The host cellmay be either prokaryotic or eukaryotic, and particularly preferred hostcells are those which express the nucleic acid segment(s) comprising therecombinant vector which encode one or more B. thuringiensis crystalprotein comprising modified amino acid sequences in one or more loopregions of domain 1, or between α helix 7 of domain 1 and β strand 1 ofdomain 2. Bacterial cells are particularly preferred as prokaryotichosts, and plant cells are particularly preferred as eukaryotic hosts

In an important embodiment, the invention discloses and claims a hostcell wherein the modified amino acid sequences comprise one or more loopregions between α helices 1 and 2, α helices 2 and 3, α helices 3 and 4,α helices 4 and 5, α helices 5 and 6 or α helices 6 and 7 of domain 1,or between α helix 7 of domain 1 and β strand 1 of domain 2. Aparticularly preferred host cell is one that comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108, and morepreferably, one that comprises the nucleic acid sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ IDNO:101, or SEQ ID NO:107.

Bacterial host cells transformed with a nucleic acid segment encoding amodified Cry3Bb crystal protein according to the present invention aredisclosed and claimed herein, and in particular, a B. thuringiensis cellhaving designation EG11221, EG11222, EG11223, EG11224, EG11225, EG11226,EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233, EG11234,EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242, EG11032,EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058, EG11081,EG11082, EG11083, EG11084, EG11095, or EG11098.

In another embodiment, the invention encompasses a method of using anucleic acid segment of the present invention that encodes a cry3Bb*gene. The method generally comprises the steps of: (a) preparing arecombinant vector in which the cry3Bb* gene is positioned under thecontrol of a promoter; (b) introducing the recombinant vector into ahost cell; (c) culturing the host cell under conditions effective toallow expression of the Cry3Bb* crystal protein encoded by said cry3Bb*gene; and (d) obtaining the expressed Cry3Bb* crystal protein orpeptide.

A wide variety of ways are available for introducing a B. thuringiensisgene expressing a toxin into the microorganism host under conditionswhich allow for stable maintenance and expression of the gene. One canprovide for DNA constructs which include the transcriptional andtranslational regulatory signals for expression of the toxin gene, thetoxin gene under their regulatory control and a DNA sequence homologouswith a sequence in the host organism, whereby integration will occur,and/or a replication system which is functional in the host, wherebyintegration or stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger RNA, particularly by using an active promoter, as well asby employing sequences, which enhance the stability of the messengerRNA. The transcriptional and translational termination region willinvolve stop codon(s), a terminator region, and optionally, apolyadenylation signal. A hydrophobic “leader” sequence may be employedat the amino terminus of the translated polypeptide sequence in order topromote secretion of the protein across the inner membrane.

In the direction of transcription, namely in the 5′ to 3′ direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5′ or 3′ of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, more preferably at least about 1000 bp, and usuallynot more than about 2000 bp of a sequence homologous with a sequence inthe host. In this way, the probability of legitimate recombination isenhanced, so that the gene will be integrated into the host and stablymaintained by the host. Desirably, the toxin gene will be in closeproximity to the gene providing for complementation as well as the geneproviding for the competitive advantage. Therefore, in the event that atoxin gene is lost, the resulting organism will be likely to also lostthe complementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the λ_(L) and λ_(R) promoters, the tac promoter, thenaturally-occurring promoters associated with the δ-endotoxin gene,where functional in the host. See for example, U.S. Pat. Nos. 4,332,898;4,342,832; and 4,356,270 (each of which is specifically incorporatedherein by reference). The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pR01614. and the like. See for example, Olson et al. (1982); Bagdasarianet al. (1981), Baum et al., 1990, and U.S. Pat. Nos. 4,356,270;4,362,817; 4,371,625, and 5,441,884, each incorporated specificallyherein by reference.

The B. thuringiensis gene can be introduced between the transcriptionaland translational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forpesticidal activity. If desired, unwanted or ancillary DNA sequences maybe selectively removed from the recombinant bacterium by employingsite-specific recombination systems, such as those described in U.S.Pat. No. 5,441,884 (specifically incorporated herein by reference).

2.4 Cry3 DNA Segments

A B. thuringiensis cry3* gene encoding a crystal protein having one ormore mutations in one or more regions of the peptide represents animportant aspect of the invention. Preferably, the cry3* gene encodes anamino acid sequence in which one or more amino acid residues have beenchanged based on the methods disclosed herein, and particularly thosechanges which have been made for the purpose of altering theinsecticidal activity or specificity of the crystal protein.

In accordance with the present invention, nucleic acid sequences includeand are not limited to DNA, including and not limited to cDNA andgenomic DNA, genes; RNA, including and not limited to mRNA and tRNA;antisense sequences, nucleosides, and suitable nucleic acid sequencessuch as those set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107, andalterations in the nucleic acid sequences including alterations,deletions, mutations, and homologs capable of expressing the B.thuringiensis modified toxins of the present invention.

As such the present invention also concerns DNA segments, that are freefrom total genomic DNA and that encode the novel synthetically-modifiedcrystal proteins disclosed herein. DNA segments encoding these peptidespecies may prove to encode proteins, polypeptides, subunits, functionaldomains, and the like of crystal protein-related or other non-relatedgene products. In addition these DNA segments may be synthesizedentirely in vitro using methods that are well-known to those of skill inthe art.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a crystal protein or peptide refers toa DNA segment that contains crystal protein coding sequences yet isisolated away from, or purified free from, total genomic DNA of thespecies from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, operonsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a gene encoding a bacterial crystalprotein, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

Particularly preferred DNA sequences are those encoding Cry3Bb.60,Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225,Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046,Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081,Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095 and Cry3Bb.11098crystal proteins, and in particular cry3Bb* genes such as cry3Bb.60,cry3Bb.11221, cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225,cry3Bb.11226, cry3Bb. 11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230,cry3Bb.11231, cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235,cry3Bb.11236, cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241,cry3Bb.11242, cry3Bb.110322 cry3Bb.11035, cry3Bb.11036, cry3Bb.11046,cry3Bb.11048, cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081,cry3Bb.11082, cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 and cry3Bb.11098.In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Crypeptide species that includes within its amino acid sequence an aminoacid sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQID NO:108.

The term “a sequence essentially as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108” means that the sequence substantiallycorresponds to a portion of the sequence of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQID NO:108, and has relatively few amino acids that are not identical to,or a biologically functional equivalent of, the amino acids of any ofthese sequences. The term “biologically functional equivalent” is wellunderstood in the art and is further defined in detail herein (e.g., seeIllustrative Embodiments).

Accordingly, sequences that have between about 70% and about 75% orbetween about 75% and about 80%, or more preferably between about 81%and about 90%, or even more preferably between about 91% or 92% or 93%and about 97% or 98% or 99% amino acid sequence identity or functionalequivalence to the amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102 or SEQ ID NO:108 willbe sequences that are “essentially as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences. and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

For example, nucleic acid fragments may be prepared that include a shortcontiguous stretch encoding the peptide sequence disclosed in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100,SEQ ID NO:102, or SEQ ID NO:108, or that are identical to orcomplementary to DNA sequences which encode the peptide disclosed in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68. SEQ ID NO:70, SEQ IDNO:100, SEQ ID NO:102, or SEQ ID NO:108, and particularly the DNAsegments disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.

Highly preferred nucleic acid segments of the present invention compriseone or more cry genes of the invention, or a portion of one or more crygenes of the invention. For certain application, relatively smallcontiguous nucleic acid sequences are preferable, such as those whichare about 14 or 15 or 16 or 17 or 18 or 19, or 20, or 30-50, 51-80,81-100 or so nucleotides in length. Alternatively, in some embodiments,and particularly those involving preparation of recombinant vectors,transformation of suitable host cells, and preparation of transgenicplant cell, longer nucleic acid segments are preferred, particularlythose that include the entire coding region of one or more cry genes. Assuch, the preferred segments may include those that are up to about20,000 or so nucleotides in length, or alternatively, shorter sequencessuch as those about 19,000, about 18,000, about 17,000, about 16,000,about 15,000, about 14,000, about 13,000, about 12,000, 11,000, about10,000, about 9,000, about 8,000, about 7,000, about 6,000, about 5,000,about 4,500, about 4,000, about 3,500, about 3,000, about 2,500, about2,000, about 1,500, about 1,000, about 500, or about 200 or so basepairs in length. Of course, these numbers are not intended to beexclusionary of all possible intermediate lengths in the range of fromabout 20,000 to about 15 nucleotides, as all of these intermediatelengths are also contemplated to be useful, and fall within the scope ofthe present invention. It will be readily understood that “intermediatelengths”, in these contexts, means any length between the quoted ranges,such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, 24, 25, 26, 27,28, 29, etc.; 30, 31, 32, 33, 34, 35, 36 . . . etc.; 40, 41, 42, 43, 44. . . etc., 50, 51, 52, 53 . . . etc.; 60, 61, 62, 63 . . . etc., 70,80, 90, 100, 110, 120, 130 . . . etc.; 200, 210, 220, 230, 240, 250 . .. etc.; including all integers in the entire range from about 14 toabout 10,000, including those integers in the ranges 200-500; 500-1,000;1,000-2,000; 2,000-3,000; 3,000-5,000 and the like.

In a preferred embodiment, the nucleic acid segments comprise a sequenceof from about 1800 to about 18,000 base pair in length, and comprise oneor more genes which encode a modified Cry3* polypeptide disclosed hereinwhich has increased activity against Coleopteran insect pests.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, or SEQ ID NO:108, including the DNA sequences which areparticularly disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107.Recombinant vectors and isolated DNA segments may therefore variouslyinclude the peptide-coding regions themselves, coding regions bearingselected alterations or modifications in the basic coding region, orthey may encode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.5 Vectors, Host Cells, and Protein Expression

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in high-level expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8, 9, 10, or 11 or soamino acids, and up to and including those of about 30, 40, or 50 or soamino acids in length, or more preferably, from about 8 to about 30amino acids in length, or even more preferably, from about 8 to about 20amino acids in length are contemplated to be particularly useful. Suchpeptide epitopes may be amino acid sequences which comprise contiguousamino acid sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:108.

2.6 Transformed Host Cells and Transgenic Plants

In one embodiment, the invention provides a transgenic plant havingincorporated into its genome a transgene that encodes a contiguous aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14. SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:100, SEQ IDNO:102, and SEQ ID NO:108.

A further aspect of the invention is a transgenic plant havingincorporated into its genome a cry3Bb* transgene, provided the transgenecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:99, SEQ ID NO:101, and SEQ ID NO:107. Also disclosed and claimed areprogeny of such a transgenic plant, as well as its seed, progeny fromsuch seeds, and seeds arising from the second and subsequent generationplants derived from such a transgenic plant.

The invention also discloses and claims host cells, both native, andgenetically engineered, which express the novel cry3Bb* genes to produceCry3Bb* polypeptides. Preferred examples of bacterial host cells includeB. thuringiensis EG11221, EG11222, EG11223, EG11224, EG11225, EG11226,EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233, EG11234,EG11235, EG11236, EG11237, EG11238, EG11239, EG11241, EG11242, EG11032,EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058, EG11081,EG11082, EG11083, EG11084, EG11095, and EG11098.

Methods of using such cells to produce Cry3* crystal proteins are alsodisclosed. Such methods generally involve culturing the host cell (suchas B. thuringiensis EG 11221, EG11222, EG11223, EG11224, EG11225,EG11226, EG11227, EG11228, EG11229, EG11230, EG11231, EG11232, EG11233,EG11234, EG11235, EG11236, EG11237. EG11238, EG11239, EG11241, EG11242,EG11032, EG11035, EG11036, EG11046, EG11048, EG11051, EG11057, EG11058,EG11081, EG11082, EG11083, EG11084, or EG11095, or EG11098) underconditions effective to produce a Cry3* crystal protein, and obtainingthe Cry3* crystal protein from said cell.

In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel recombinant crystal proteins of the presentinvention. The process of producing transgenic plants is well-known inthe art. In general, the method comprises transforming a suitable hostcell with one or more DNA segments which contain one or more promotersoperatively linked to a coding region that encodes one or more of thedisclosed B. thuringiensis crystal proteins. Such a coding region isgenerally operatively linked to a transcription-terminating region,whereby the promoter is capable of driving the transcription of thecoding region in the cell, and hence providing the cell the ability toproduce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

Another aspect of the invention comprises a transgenic plant whichexpress a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression.

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of one or more Cry3Bb*-encoding transgenes, eithernative, synthetically modified, or mutated. In some instances, more thanone transgene will be incorporated into the genome of the transformedhost plant cell. Such is the case when more than one crystalprotein-encoding DNA segment is incorporated into the genome of such aplant. In certain situations, it may be desirable to have one, two,three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding a DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art, and are discussed herein. Vectors,plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segmentsfor use in transforming such cells will, of course, generally compriseeither the operons, genes, or gene-derived sequences of the presentinvention, either native, or synthetically-derived, and particularlythose encoding the disclosed crystal proteins. These DNA constructs canfurther include structures such as promoters, enhancers, polylinkers, oreven gene sequences which have positively- or negatively-regulatingactivity upon the particular genes of interest as desired. The DNAsegment or gene may encode either a native or modified crystal protein,which will be expressed in the resultant recombinant cells, and/or whichwill impart an improved phenotype to the regenerated plant

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encoding aCry3Bb* crystal protein which is toxic to coleopteran insects.Particularly preferred plants include grains such as corn, wheat, rye,rice, barley, and oats; legumes such as soybeans; tubers such aspotatoes; fiber crops such as flax and cotton; turf and pasture grasses;ornamental plants; shrubs; trees; vegetables, berries, citrus, fruits,cacti, succulents, and other commercially-important crops includinggarden and houseplants.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have oneor more crystal protein transgene(s) stably incorporated into itsgenome, and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more Cry3Bb* crystal proteins or polypeptidesare aspects of this invention. Particularly preferred transgenes for thepractice of the invention include nucleic acid segments comprising oneor more cry3Bb* gene(s).

2.7 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 4.

TABLE 4 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG GUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, specifically incorporated herein by reference, statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. Schematic representation of the monomeric structure of Cry3Bb.

FIG. 2. Stereoscopic view of the monomeric structure of Cry3Bb withassociated water molecules (represented by dots).

FIG. 3A. Schematic representation of domain 1 of Cry3Bb

FIG. 3B. Diagram of the positions of the 7 helices that comprise domain1.

FIG. 4. Domain 1 of Cry3Bb is organized into seven α helices illustratedin FIG. 3A (schematic representation) and FIG. 3B (schematic diagram).The α helices and amino acids residues are shown.

FIG. 5A. Schematic representation of domain 2 of Cry3Bb.

FIG. 5B. Diagram of the positions of the 11 β strands that compose the 3βsheets of domain 2.

FIG. 6. Domain 2 of Cry3Bb is a collection of three anti-parallel βsheets illustrated in FIG. 5. The amino acids that define these sheetsis listed below (α8, amino aids 322-328, also is included in domain 2):

FIG. 7A. Schematic representation of domain 3 of Cry3Bb.

FIG. 7B. Diagram of the positions of the β strands that comprise domain3.

FIG. 8. Domain 3 (FIG. 7) is a loosely organized collection of β strandsand loops; no β sheets are present. The β stands contain the amino acidslimited below:

FIG. 9A. A “side” view of the dimeric structure of Cry3Bb. The helicalbundles of domains 1 can be seem in the middle of the molecule.

FIG. 9B. A “top” view of the dimeric structure of Cry3Bb. The helicalbundles of domains 1 can be seem in the middle of the molecule.

FIG. 10. A graphic representation of the growth in conductance with timeof channels formed by Cry3A and Cry3Bb in planar lipid bilayers. Cry3Aforms channels with higher conductances much more rapidly than Cry3Bb.

FIG. 11. A map of pEG1701 which contains the Cry3Bb gene with the cry1Fterminator.

FIG. 12. The results of replicated 1-dose assays against SCRW larvae ofCry3Bb proteins altered in the 1B2,3 region.

FIG. 13. The results of replicated, 1-dose assays against SCRW larvae ofCry3Bb proteins altered in the 1B6, 7 region.

FIG. 14. The results of replicated, 1-dose screens against SCRW larvaeof Cry3Bb proteins altered in the 1B10,11 region.

FIG. 15. Single channel recordings of channels formed by Cry3Bb.11230and WT Cry3Bb in planar lipid bilayers. Cry3Bb.11230 forms channels withwell resolved open and closed states while Cry3Bb rarely does.

FIG. 16. Single channel recordings of channels formed by Cry3Bb andCry3Bb.60, a truzncated form of Cry3Bb. Cry3Bb.60 forms channels morequickly than Cry3Bb and, unlike Cry3Bb, produces channels with wellresolved open and closed states.

FIG. 17A. Sequence alignment of the amino acid sequences of Cry3C,Cry3BB2, Cry3BB, Cry3BA and Cry3A.

FIG. 17B. Shown is a continuation of alignment of the amino acidsequences of Cry3C, Cry3BB2, Cry3BB, Cry3BA and Cry3A shown in FIG. 17A.

FIG. 17C. Shown is a continuation of alignment of the amino acidsequences of Cry3C (SEQ ID NO:109), Cry3BB2 (SEQ ID NO:110), Cry3BB (SEQID NO:111), Cry3BA (SEQ ID NO:112) and Cry3A (SEQ ID NO:113) shown inFIG. 17A.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention defines new B. thuringiensis (Bt) insecticidal δ-endotoxinproteins and the biochemical and biophysical strategies used to designthe new proteins. Delta-endotoxins are a class of insecticdal proteinsproduced by B. thuringiensis that form cation-selective channels inplanar lipid bilayers (English and Slatin, 1992). The new δ-endotoxinsare based on the parent structure of the coleopteran-active, δ-endotoxinCry3Bb. Like other members of the coleopteran-active class ofδ-endotoxins, including Cry3A and Cry3B, Cry3Bb exhibits excellentinsecticidal activity against the Colorado Potato Beetle (Leptinotarsadecemlineata). However, unlike Cry3A and Cry3B, Cry3Bb is also activeagainst the southern corn rootworm or SCRW (Diabrotica undecimpunctatahowardi Barber) and the western corn rootworm or WCRW (Diabroticavirgifera virgifera LeConte). The new insecticidal proteins describedherein were specifically designed to improve the biological activity ofthe parent Cry3Bb protein. In addition, the design strategies themselvesare novel inventions capable of being applied to and improving B.thuringiensis δ-endotoxins in general. B. thuringiensis δ-endotoxins arealso members of a larger class of bacterial toxins that form ionchannels (see English and Slatin 1992, for a review). The inventors,therefore, believe that these design strategies can also be applied toany biologically active, channel-forming protein to improve itsbiological properties.

The designed Cry3Bb proteins were engineered using one or more of thefollowing strategies including (1) identification and alteration ofprotease-sensitive sites and proteolytic processing; (2) analysis andmanipulation of bound water; (3) manipulation of hydrogen bonds aroundmobile regions; (4) loop analysis and loop redesign around flexiblehelices; (5) loop design around β strands and β sheets; (6)identification and redesign of complex electrostatic surfaces; (7)identification and removal of metal binding sites; (8) alteration ofquaternary structure; (9) identification and design of structuralresidues; and (10) combinations of any and all sites defined bystrategies 1-9. These design strategies permit the identification andredesign of specific sites on Cry3Bb, ultimately creating new proteinswith improved insecticidal activities. These new proteins are designatedCry3Bb designed proteins and are named Cry3Bb followed by a period and asuffix (e.g., Cry3Bb.60, Cry3Bb.11231). The new proteins are listed inTable 2 along with the specific sites on the molecule that weremodified, the amino-acid sequence changes at those sites that improvebiological activity, the improved insecticidal activities and the designmethod used to identify that specific site.

4.1 Some Advantages of the Invention

Mutagenesis studies with cry genes have failed to identify a significantnumber of mutant crystal proteins which have improved broad-spectruminsecticidal activity, that is, with improved toxicity towards a rangeof insect pest species. Since agricultural crops are typicallythreatened by more than one insect pest species at any given time,desirable mutant crystal proteins are preferably those that exhibitimprovements in toxicity towards multiple insect pest species. Previousfailures to identify such mutants may be attributed to the choice ofsites targeted for mutagenesis. For example, with respect to the relatedprotein, Cry1C, sites within domain 2 and domain 3 have been theprincipal targets of mutagenesis efforts, primarily because thesedomains are believed to be important for receptor binding and indetermining insecticidal specificity (Aronson et al., 1995; Chen et al.1993; de Maagd et al., 1996; Lee et al., 1992; Lee et al., 1995; Lu etal., 1994; Smedley and Ellar, 1996; Smith and Ellar, 1994; Rajamohan etal., 1995; Rajamohan et al., 1996)

In contrast, the present inventors reasoned that the toxicity of Cry3proteins, and specifically the toxicity of the Cry3Bb protein, may beimproved against a broader array of target pests by targeting regionsinvolved in ion channel function rather than regions of the moleculedirectly involved in receptor interactions, namely domains 2 and 3.Accordingly, the inventors opted to target regions within domain 1 ofCry3Bb for mutagenesis for the purpose of isolating Cry3Bb mutants withimproved broad spectrum toxicity. Indeed, in the present invention,Cry3Bb mutants are described that show improved toxicity towards severalcoleopteran pests.

At least one, and probably more than one, α helix of domain 1 isinvolved in the formation of ion channels and pores within the insectmidgut epithelium (Gazit and Shai, 1993; Gazit and Shai, 1995). Ratherthan target for mutagenesis the sequences encoding the α helices ofdomain 1 as others have (Wu and Aronson, 1992; Aronson et al., 1995;Chen et al., 1995), the present inventors opted to target exclusivelysequences encoding amino acid residues adjacent to or lying within thepredicted loop regions of Cry3Bb that separate these α helices. Aminoacid residues within these loop regions or amino acid residues cappingthe end of an α helix and lying adjacent to these loop regions mayaffect the spatial relationships among these α helices. Consequently,the substitution of these amino acid residues may result in subtlechanges in tertiary structure, or even quaternary structure, thatpositively impact the function of the ion channel. Amino acid residuesin the loop regions of domain 1 are exposed to the solvent and thus areavailable for various molecular interactions. Altering these amino acidscould result in greater stability of the protein by eliminating oroccluding protease-sensitive sites. Amino acid substitutions that changethe surface charge of domain 1 could alter ion channel efficiency oralter interactions with the brush border membrane or with other portionsof the toxin molecule, allowing binding or insertion to be moreeffective.

According to this invention, base substitutions are made in theunderlying cry3Bb nucleic acid residues in order to change particularcodons of the corresponding polypeptides, and particularly, in thoseloop regions between α-helices. The insecticidal activity of a crystalprotein ultimately dictates the level of crystal protein required foreffective insect control. The potency of an insecticidal protein shouldbe maximized as much as possible in order to provide for its economicand efficient utilization in the field. The increased potency of aninsecticidal protein in a bioinsecticide formulation would be expectedto improve the field performance of the bioinsecticide product.Alternatively, increased potency of an insecticidal protein in abioinsecticide formulation may promote use of reduced amounts ofbioinsecticide per unit area of treated crop, thereby allowing for morecost-effective use of the bioinsecticide product. When expressed inplanta, the production of crystal proteins with improved insecticidalactivity can be expected to improve plant resistance to susceptibleinsect pests.

4.2 Methods for Culturing B. thuringiensis to Produce Crystal Proteins

The B. thuringiensis strains described herein may be cultured usingstandard known media and fermentation techniques. Upon completion of thefermentation cycle, the bacteria may be harvested by first separatingthe B. thuringiensis spores and crystals from the fermentation broth bymeans well known in the art. The recovered B. thuringiensis spores andcrystals can be formulated into a wettable powder, a liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. The formulation andapplication procedures are all well known in the art.

4.3 Recombinant Host Cells for Expression of Cry* Genes

The nucleotide sequences of the subject invention can be introduced intoa wide variety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the sites of coleopteran insects where they willproliferate and be ingested by the insects. The result is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B. thuringiensis toxin.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility or toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B. thuringiensisgene into the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide micro-capsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; leaf affinity; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, B. thuringiensis, Escherichiacoli, B. subtilis, B. megaterium, B. cereus, Streptomyces lividans andthe like.

Treatment of the microbial cell, e. g., a microbe containing the B.thuringiensis toxin gene, can be by chemical or physical means, or by acombination of chemical and/or physical means, so long as the techniquedoes not deleteriously affect the properties of the toxin, nor diminishthe cellular capability in protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehye; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Lugol's iodine, Bouin's fixative,and Helly's fixatives, (see e.g., Humason, 1967); or a combination ofphysical (heat) and chemical agents that preserve and prolong theactivity of the toxin produced in the cell when the cell is administeredto the host animal. Examples of physical means are short wavelengthradiation such as γ-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. The cells employed will usually be intactand be substantially in the proliferative form when treated, rather thanin a spore form, although in some instances spores may be employed.

Where the B. thuringiensis toxin gene is introduced via a suitablevector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus(including the species and subspecies B. thuringiensis kurstaki HD-1, B.thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensisberliner, B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B.thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensisgalleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.thuringiensis entomocidus, B. thuringiensis tenebrionis and B.thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobactersphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans.

4.4 Definitions

In accordance with the present invention, nucleic acid sequences includeand are not limited to DNA (including and not limited to genomic orextragenomic DNA), genes, RNA (including and not limited to mRNA andtRNA), nucleosides, and suitable nucleic acid segments either obtainedfrom native sources, chemically synthesized, modified, or otherwiseprepared by the hand of man. The following words and phrases have themeanings set forth below.

A, an: In accordance with long standing patent law convention, the words“a” and “an” when used in this application, including the claims,denotes “one or more”.

Broad-spectrum: Refers to a wide range of insect species.

Broad-spectrum activity: The toxicity towards a wide range of insectspecies.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Insecticidal activity: The toxicity towards insects.

Insecticidal specificity: The toxicity exhibited by a crystal protein orproteins, microbe or plant, towards multiple insect species.

Intraorder specificity: The toxicity of a particular crystal proteintowards insect species within an Order of insects (e.g., OrderColeoptera).

Interorder specificity: The toxicity of a particular crystal proteintowards insect species of different Orders (e.g., Orders Coleoptera andDiptera).

LC₅₀: The lethal concentration of crystal protein that causes 50%mortality of the insects treated.

LC₉₅: The lethal concentration of crystal protein that causes 95%mortality of the insects treated.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular cellssuch as leaf, root, stem, e.g., somatic cells, or reproductive (germ)cells obtained from a transgenic plant.

Transgenic plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

As used herein, the designations “CryIII” and “Cry3” are synonymous, asare the designations “CryIIIB2” and “Cry3Bb.” Likewise, the inventorshave utilized the generic term Cry3Bb* to denote any and all Cry3Bbvariants which comprise amino acid sequences modified in the protein.Similarly, cry3Bb* is meant to denote any and all nucleic acid segmentsand/or genes which encode a Cry3Bb* protein, etc.

4.5 Preparation of Cry3* Polynucleotides

Once the structure of the desired peptide to be mutagenized has beenanalyzed using one or more of the design strategies disclosed herein, itwill be desirable to introduce one or more mutations into either theprotein or, alternatively, into the DNA sequence encoding the proteinfor the purpose of producing a mutated protein with alteredbioinsecticidal properties.

To that end, the present invention encompasses both site-specificmutagenesis methods and random mutagenesis of a nucleic acid segmentencoding a crystal protein in the manner described herein. Inparticular, methods are disclosed for the mutagenesis of nucleic acidsegments encoding the amino acid sequences using one or more of thedesign strategies described herein. Using the assay methods describedherein, one may then identify mutants arising from these procedureswhich have improved insecticidal properties or altered specificity,either intraorder or interorder.

The means for mutagenizing a DNA segment encoding a crystal protein arewell-known to those of skill in the art. Modifications may be made byrandom, or site-specific mutagenesis procedures. The nucleic acid may bemodified by altering its structure through the addition or deletion ofone or more nucleotides from the sequence.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art such as and not limited to synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular crystal protein. A “suitable host” is any host which willexpress Cry3Bb, such as and not limited to B. thuringiensis and E. coli.Screening for insecticidal activity, in the case of Cry3Bb includes andis not limited to coleopteran-toxic activity which may be screened forby techniques known in the art.

In particular, site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the DNA. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 17 to about 75 nucleotides ormore in length is preferred, with about 10 to about 25 or more residueson both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform or transfect appropriate cells, such as E. colicells, and clones are selected which include recombinant vectors bearingthe mutated sequence arrangement. A genetic selection scheme was devisedby Kunkel et al. (1987) to enrich for clones incorporating the mutagenicoligonucleotide. Alternatively, the use of PCR™ with commerciallyavailable thermostable enzymes such as Taq polymerase may be used toincorporate a mutagenic oligonucleotide primer into an amplified DNAfragment that can then be cloned into an appropriate cloning orexpression vector. The PCR™-mediated mutagenesis procedures of Tomic etal. (1990) and Upender et al. (1995) provide two examples of suchprotocols. A PCR™ employing a thermostable ligase in addition to athermostable polymerase may also be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994) provides an example ofone such protocol.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159(each of which is specifically incorporated herein by reference in itsentirety). Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction products and the process isrepeated. Preferably a reverse transcriptase PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308, incorporatedherein by reference in its entirety. In LCR, two complementary probepairs are prepared, and in the presence of the target sequence, eachpair will bind to opposite complementary strands of the target such thatthey abut. In the presence of a ligase, the two probe pairs will link toform a single unit. By temperature cycling, as in PCR™, bound ligatedunits dissociate from the target and then serve as “target sequences”for ligation of excess probe pairs. U.S. Pat. No. 4,883,750,specifically incorporated herein by reference in its entirety, describesan alternative method of amplification similar to LCR for binding probepairs to a target sequence.

Qbeta Replicase™, described in Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA which has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site (Walker et al., 1992, incorporated herein by referencein its entirety), may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA

Sequences can also be detected using a cyclic probe reaction (CPR). InCPR, a probe having 3′ and 5′ end sequences of non-Cry-specific DNA andan internal sequence of a Cry-specific RNA is hybridized to DNA which ispresent in a sample. Upon hybridization, the reaction is treated withRNaseH, and the products of the probe identified as distinctive productsgenerating a signal which are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated. Thus, CPR involves amplifying a signal generated byhybridization of a probe to a cry-specific expressed nucleic acid

Still other amplification methods described in Great Britain Pat. Appl.No. 2 202 328, and in Intl. Pat. Appl. Publ. No. PCT/US89/01025, each ofwhich is incorporated herein by reference in its entirety, may be usedin accordance with the present invention. In the former application,“modified” primers are used in a PCR™ like, template and enzymedependent synthesis. The primers may be modified by labeling with acapture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh et al., 1989; Intl. Pat. Appl. Publ.No. WO 88/10315, incorporated herein by reference in its entirety),including nucleic acid sequence based amplification (NASBA) and 3SR. InNASBA, the nucleic acids can be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a sample, treatmentwith lysis buffer and minispin columns for isolation of DNA and RNA orguanidinium chloride extraction of RNA. These amplification techniquesinvolve annealing a primer which has crystal protein-specific sequences.Following polymerization, DNA/RNA hybrids are digested with RNase Hwhile double stranded DNA molecules are heat denatured again. In eithercase the single stranded DNA is made fully double stranded by additionof second crystal protein-specific primer, followed by polymerization.The double stranded DNA molecules are then multiply transcribed by apolymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAsare reverse transcribed into double stranded DNA, and transcribed onceagainst with a polymerase such as T7 or SP6. The resulting products,whether truncated or complete, indicate crystal protein-specificsequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference inits entirety, disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA

Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by referencein its entirety, disclose a nucleic acid sequence amplification schemebased on the hybridization of a promoter/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic; i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” (Frohman, 1990), and “one-sided PCR™” (Ohara,1989) which are well-known to those of skill in the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

4.6 Phage-Resistant Variants

In certain embodiments, one may desired to prepare one or more phageresistant variants of the B. thuringiensis mutants prepared by themethods described herein. To do so, an aliquot of a phage lysate isspread onto nutrient agar and allowed to dry. An aliquot of the phagesensitive bacterial strain is then plated directly over the dried lysateand allowed to dry. The plates are incubated at 30° C. The plates areincubated for 2 days and, at that time, numerous colonies could be seengrowing on the agar. Some of these colonies are picked and subculturedonto nutrient agar plates. These apparent resistant cultures are testedfor resistance by cross streaking with the phage lysate. A line of thephage lysate is streaked on the plate and allowed to dry. Thepresumptive resistant cultures are then streaked across the phage line.Resistant bacterial cultures show no lysis anywhere in the streak acrossthe phage line after overnight incubation at 30° C. The resistance tophage is then reconfirmed by plating a lawn of the resistant cultureonto a nutrient agar plate. The sensitive strain is also plated in thesame manner to serve as the positive control. After drying, a drop ofthe phage lysate is plated in the center of the plate and allowed todry. Resistant cultures showed no lysis in the area where the phagelysate has been placed after incubation at 30° C. for 24 hours.

4.7 Crystal Protein Compositions as Insecticides and Methods of Use

Order Coleoptera comprises numerous beetle species including groundbeetles, reticulated beetles, skin and larder beetles, long-hornedbeetles, leaf beetles, weevils, bark beetles, ladybird beetles, soldierbeetles, stag beetles, water scavenger beetles, and a host of otherbeetles. A brief taxonomy of the Order is given at the websitehttp://www.ncbi.nlm.nih.gov/Taxonomy/tax.html.

Particularly important among the Coleoptera are the agricultural pestsincluded within the infraorders Chrysomeliformia and Cucujiformia.Members of the infraorder Chrysomeliformia, including the leaf beetles(Chrysomelidae) and the weevils (Curculionidae), are particularlyproblematic to agriculture, and are responsible for a variety of insectdamage to crops and plants. The infraorder Cucujiformia includes thefamilies Coccinellidae, Cucujidae, Lagridae, Meloidae, Rhipiphoridae,and Tenebrionidae. Within this infraorder, members of the familyChrysomelidae (which includes the genera Exema, Chrysomela, Oreina,Chrysolina, Leptinotarsa, Gonioctena, Oulema, Monozia, Ophraella,Cerotoma, Diabrotica, and Lachnaia), are well-known for their potentialto destroy agricultural crops.

As the toxins of the present invention have been shown to be effectivein combatting a variety of members of the order Coleoptera, theinventors contemplate that the insects of many Coleopteran genera may becontrolled or eradicated using the polypeptide compositions describedherein. Likewise, the methods described herein for generating modifiedpolypeptides having enhanced insect specificity may also be useful inextending the range of the insecticidal activity of the modifiedpolypeptides to other insect species within, and outside of, the OrderColeoptera.

As such, the inventors contemplate that the crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, including but notlimited to rice, wheat, alfalfa, corn (maize), soybeans, tobacco,potato, barley, canola (rapeseed), sugarbeet, sugarcane, flax, rye,oats, cotton, sunflower; grasses, such as pasture and turf grasses;fruits, citrus, nuts, trees, shrubs and vegetables; as well asornamental plants, cacti, succulents, and the like.

Disclosed and claimed is a composition comprising aninsecticidally-effective amount of a Cry3Bb* crystal proteincomposition. The composition preferably comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:100, or SEQ ID NO:108 or biologically-functionalequivalents thereof.

The insecticide composition may also comprise a Cry3Bb* crystal proteinthat is encoded by a nucleic acid sequence having the sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NQ:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, orSEQ ID NO:108, or, alternatively, a nucleic acid sequence whichhybridizes to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, or SEQ ID NO:107 underconditions of moderate stringency.

The insecticidal compositions may comprise one or more B. thuringiensiscell types, or one or more cultures of such cells, or, alternatively, amixture of one or more B. thuringiensis cells which express one or moreof the novel crystal proteins of the invention in combination withanother insecticidal composition. In certain aspects it may be desirableto prepare compositions which contain a plurality of crystal proteins,either native or modified, for treatment of one or more types ofsusceptible insects. The B. thuringiensis cells of the invention can betreated prior to formulation to prolong the insecticidal activity whenthe cells are applied to the environment of the target insect(s). Suchtreatment can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the insecticide, nor diminish thecellular capability in protecting the insecticide. Examples of chemicalreagents are halogenerating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehyde; anti-infectives, such as zephiran chloride; alcohols,such as isopropyl and ethanol; various histologic fixatives, such asBouin's fixative and Helly's fixative (see Humason, 1967); or acombination of physical (heat) and chemical agents that prolong theactivity of the δ-endotoxin produced in the cell when the cell isapplied to the environment of the target pest(s). Examples of physicalmeans are short wavelength radiation such as gamma-radiation andX-radiation, freezing, UV irradiation, lyophilization, and the like.

The inventors contemplate that any formulation methods known to those ofskill in the art may be employed using the proteins disclosed herein toprepare such bioinsecticide compositions. It may be desirable toformulate whole cell preparations, cell extracts, cell suspensions, cellhomogenates, cell lysates, cell supernatants, cell filtrates, or cellpellets of a cell culture (preferably a bacterial cell culture such as aB. thuringiensis cell culture described in Table 3) that expresses oneor more cry3Bb* DNA segments to produce the encoded Cry3Bb* protein(s)or peptide(s). The methods for preparing such formulations are known tothose of skill in the art, and may include, e.g., desiccation,lyophilization, homogenization, extraction, filtration, centrifugation,sedimentation, or concentration of one or more cultures of bacterialcells, such as B. thuringiensis cells described in Table 3, whichexpress the Cry3Bb* peptide(s) of interest.

In one preferred embodiment, the bioinsecticide composition comprises anoil flowable suspension comprising lysed or unlysed bacterial cells,spores, or crystals which contain one or more of the novel crystalproteins disclosed herein. Preferably the cells are B. thuringiensiscells, however, any such bacterial host cell expressing the novelnucleic acid segments disclosed herein and producing a crystal proteinis contemplated to be useful, such as Bacillus spp., including B.megaterium, B. subtilis; B. cereus, Escherichia spp., including E. coli,and/or Pseudomonas spp., including P. cepacia, P. aeruginosa, and P.fluorescens. Alternatively, the oil flowable suspension may consist of acombination of one or more of the following compositions: lysed orunlysed bacterial cells, spores, crystals, and/or purified crystalproteins.

In a second preferred embodiment, the bioinsecticide compositioncomprises a water dispersible granule or powder. This granule or powdermay comprise lysed or unlysed bacterial cells, spores, or crystals whichcontain one or more of the novel crystal proteins disclosed herein.Preferred sources for these compositions include bacterial cells such asB. thuringiensis cells, however, bacteria of the genera Bacillus,Escherichia, and Pseudomonas which have been transformed with a DNAsegment disclosed herein and expressing the crystal protein are alsocontemplated to be useful. Alternatively, the granule or powder mayconsist of a combination of one or more of the following compositions:lysed or unlysed bacterial cells, spores, crystals, and/or purifiedcrystal proteins.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, spray, emulsion, colloid, aqueous ororganic solution, dust, pellet, or collodial concentrate. Such acomposition may contain either unlysed or lysed bacterial cells, spores,crystals, or cell extracts as described above, which contain one or moreof the novel crystal proteins disclosed herein. Preferred bacterialcells are B. thuringiensis cells, however, bacteria such as B.megaterium, B. subtilis, B. cereus, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful. Such dry forms ofthe insecticidal compositions may be formulated to dissolve immediatelyupon wetting, or alternatively, dissolve in a controlled-release,sustained-release, or other time-dependent manner. Alternatively, such acomposition may consist of a combination of one or more of the followingcompositions: lysed or unlysed bacterial cells, spores, crystals, and/orpurified crystal proteins.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous solution or suspension or cell culture of lysed orunlysed bacterial cells, spores, crystals, or a mixture of lysed orunlysed bacterial cells, spores, and/or crystals, such as thosedescribed above which contain one or more of the novel crystal proteinsdisclosed herein. Such aqueous solutions or suspensions may be providedas a concentrated stock solution which is diluted prior to application,or alternatively, as a diluted solution ready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the Crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise B. thuringiensis cells,spores, and/or crystals containing the modified crystal protein(s) ofinterest, such compositions may be formulated in a variety of ways. Theymay be employed as wettable powders, granules or dusts, by mixing withvarious inert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel Cry3Bb-derived mutated crystal proteins may beprepared by native or recombinant bacterial expression systems in vitroand isolated for subsequent field application. Such protein may beeither in crude cell lysates, suspensions, colloids, etc., oralternatively may be purified, refined, buffered, and/or furtherprocessed, before formulating in an active biocidal formulation.Likewise, under certain circumstances, it may be desirable to isolatecrystals and/or spores from bacterial cultures expressing the crystalprotein and apply solutions, suspensions, or collodial preparations ofsuch crystals and/or spores as the active bioinsecticidal composition.

Another important aspect of the invention is a method of controllingcoleopteran insects which are susceptible to the novel compositionsdisclosed herein. Such a method generally comprises contacting theinsect or insect population, colony, etc., with aninsecticidally-effective amount of a Cry3Bb* crystal proteincomposition. The method may utilize Cry3Bb* crystal proteins such asthose disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, SEQ ID NO:100, or SEQ ID NO:108, or biologicallyfunctional equivalents thereof.

Alternatively, the method may utilize one or more Cry3Bb* crystalproteins which are encoded by the nucleic acid sequences of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ IDNO:101, or SEQ ID NO:107, or by one or more nucleic acid sequences whichhybridize to the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5. SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13. SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:107,under conditions of moderate, or higher, stringency. The methods foridentifying sequences which hybridize to those disclosed underconditions of moderate or higher stringency are well-known to those ofskill in the art, and are discussed herein.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, soil tilling, seed coating, seedling coating, spraying,aerating, misting, atomizing, and the like, are also feasible and may berequired under certain circumstances such as e.g., insects that causeroot or stalk infestation, or for application to delicate vegetation orornamental plants. These application procedures are also well-known tothose of skill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 1g to about 1 kg, 2 kg, 5, kg, or more of active ingredient.

4.8 Nucleic Acid Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99,SEQ ID NO:101, or SEQ ID NO:107 will find particular utility. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (includingall intermediate lengths and up to and including full-length sequenceswill also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to DNA sequences of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13. SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:99,SEQ ID NO:101, or SEQ ID NO:107 are particularly contemplated ashybridization probes for use in, e.g., Southern and Northern blotting.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 10-14 and about 100 or 200 nucleotides, but largercontiguous complementary stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

The use of a hybridization probe of about 14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1994; Segal 1976; Prokop, 1991; and Kuby, 1994, are particularlyrelevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

4.9 Characters of Modified Cry3 δ-Endotoxins

The present invention provides novel polypeptides that define a whole ora portion of a B. thuringiensis cry3Bb.60, cry3Bb.11221, cry3Bb.11222,cry3Bb.11223, cry3Bb.11224, cry3Bb.11225, cry3Bb.11226, cry3Bb.11227,cry3Bb.11228, cry3Bb.11229, cry3Bb.11230, cry3Bb.11231, cry3Bb.11232,cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236, cry3Bb.11237,cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242, cry3Bb.11032,cry3Bb.11035, cry3Bb.11036, cry3Bb.11046, cry3Bb.11048, cry3Bb.11051,cry3Bb.11057, cry3Bb.11058, cry3Bb.11081, cry3Bb.11082, cry3Bb.11083,cry3Bb.11084, cry3Bb.11095 and cry3Bb.11098-encoded crystal protein.

4.10 Crystal Protein Nomenclature

The inventors have arbitrarily assigned the designations Cry3Bb.60,Cry3Bb.11221, Cry3Bb.11222, Cry3Bb.11223, Cry3Bb.11224, Cry3Bb.11225,Cry3Bb.11226, Cry3Bb.11227, Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11231, Cry3Bb.11232, Cry3Bb.11233, Cry3Bb.11234, Cry3Bb.11235,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, Cry3Bb.11239, Cry3Bb.11241,Cry3Bb.11242, Cry3Bb.11032, Cry3Bb.11035, Cry3Bb.11036, Cry3Bb.11046,Cry3Bb.11048, Cry3Bb.11051, Cry3Bb.11057, Cry3Bb.11058, Cry3Bb.11081,Cry3Bb.11082, Cry3Bb.11083, Cry3Bb.11084, Cry3Bb.11095 and Cry3Bb.11098to the novel proteins of the invention.

Likewise, the arbitrary designations of cry3Bb.60, cry3Bb.11221,cry3Bb.11222, cry3Bb.11223, cry3Bb.11224, cry3Bb.11225, cry3Bb.11226,cry3Bb.11227, cry3Bb.11228, cry3Bb.11229, cry3Bb.11230, cry3Bb.11231,cry3Bb.11232, cry3Bb.11233, cry3Bb.11234, cry3Bb.11235, cry3Bb.11236,cry3Bb.11237, cry3Bb.11238, cry3Bb.11239, cry3Bb.11241, cry3Bb.11242,cry3Bb.11032, cry3Bb.11035, cry3Bb.11036, cry3Bb.11046, cry3Bb.11048,cry3Bb.11051, cry3Bb.11057, cry3Bb.11058, cry3Bb.11081, cry3Bb.11082,cry3Bb.11083, cry3Bb.11084, cry3Bb.11095 and Cry3Bb.11098 have beenassigned to the novel nucleic acid sequences which encode thesepolypeptides, respectively. While formal assignment of gene and proteindesignations based on the revised nomenclature of crystal proteinendotoxins (Table 1) may be made by the committee on the nomenclature ofB. thuringiensis, any re-designations of the compositions of the presentinvention are also contemplated to be fully within the scope of thepresent disclosure.

4.11 Transformed Host Cells and Transgenic Plants

A bacterium, a yeast cell, or a plant cell or a plant transformed withan expression vector of the present invention is also contemplated. Atransgenic bacterium, yeast cell, plant cell or plant derived from sucha transformed or transgenic cell is also one aspect of the invention.

Such transformed host cells are often desirable for use in theproduction of endotoxins and for expression of the various DNA geneconstrcuts disclosed herein. In some aspects of the invention, it isoften desirable to modulate, regulate, or otherwise control theexpression of the gene segments disclosed herein. Such methods areroutine to those of skill in the molecular genetic arts. Typically, whenincreased or over-expression of a particular gene is desired, variousmanipulations may be employed for enhancing the expression of themessenger RNA, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA inthe particular transformed host cell.

Typically, the initiation and translational termination region willinvolve stop codon(s), a terminator region, and optionally, apolyadenylation signal. In the direction of transcription, namely in the5′ to 3′ direction of the coding or sense sequence, the construct willinvolve the transcriptional regulatory region, if any, and the promoter,where the regulatory region may be either 5′ or 3′ of the promoter, theribosomal binding site, the initiation codon, the structural gene havingan open reading frame in phase with the initiation codon, the stopcodon(s), the polyadenylation signal sequence, if any, and theterminator region. This sequence as a double strand may be used byitself for transformation of a microorganism host, but will usually beincluded with a DNA sequence involving a marker, where the second DNAsequence may be joined to the δ-endotoxin expression construct duringintroduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the δ-endotoxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the δ-endotoxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the δ-endotoxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a δ-endotoxin geneis lost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

The crystal protein-encoding gene can be introduced between thetranscriptional and translational initiation region and thetranscriptional and translational termination region, so as to be underthe regulatory control of the initiation region. This construct will beincluded in a plasmid, which will include at least one replicationsystem, but may include more than one, where one replication system isemployed for cloning during the development of the plasmid and thesecond replication system is necessary for functioning in the ultimatehost. In addition, one or more markers may be present, which have beendescribed previously. Where integration is desired, the plasmid willdesirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms when present. The transformants then can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the δ-endotoxin in the cell when thethen treated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the δ-endotoxin is unstable or thelevel of application sufficiently low as to avoid any possibility oftoxicity to a mammalian host. As hosts, of particular interest will bethe prokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobitim; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibdo, Spirillum; Lactobacillaceae; phylloplane organisms such asmembers of the Pseudomonadaceae (including Pseudomonas spp. andAcetobacter spp.); Azotobacteraceae and Nitrobacteraceae; Flavobacteriumspp.; members of the Bacillaceae such as Lactobacillus spp.,Bifidobacterium, and Bacillus spp., and the like. Particularly preferredhost cells include Pseudomonas aeruginosa, Pseudomonas fluorescens,Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and thelike.

Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Schizosaccharomyces; and Basidiomycetes,Rhodotorula, Aureobasidium, Sporobolomyces, Saccharomyces spp., andSporobolomyces spp.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the δ-endotoxin geneinto the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide micro-capsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; leaf affinity; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the δ-endotoxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed. Treatment of the recombinantmicrobial cell can be done as disclosed infra. The treated cellsgenerally will have enhanced structural stability which will enhanceresistance to environmental conditions.

Genes or other nucleic acid segments, as disclosed herein, can beinserted into host cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher organisms, including plants. Thevectors comprise, for example, pBR322, pUC series, M13mp series,pACYC184, etc. Accordingly, the sequence coding for the δ-endotoxin canbe inserted into the vector at a suitable restriction site. Theresulting plasmid is used for transformation into E. coli. The E. colicells are cultivated in a suitable nutrient medium, then harvested andlysed. The plasmid is recovered. Sequence analysis, restrictionanalysis, electrophoresis, and other biochemical-molecular biologicalmethods are generally carried out as methods of analysis. After eachmanipulation, the DNA sequence used can be cleaved and joined to thenext DNA sequence. Each plasmid sequence can be cloned in the same orother plasmids. Depending on the method of inserting desired genes intothe plant, other DNA sequences may be necessary.

Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al.,1985) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993);(3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson,1988; Eglitis et al., 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1991; 1992; Wagner et al., 1992).

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobactedum rhizogenes astransformation agent, fusion, injection, or electroporation as well asother possible methods. If agrobacteria are used for the transformation,the DNA to be inserted has to be cloned into special plasmids, namelyeither into an intermediate vector or into a binary vector. Theintermediate vectors can be integrated into the Ti or Ri plasmid byhomologous recombination owing to sequences that are homologous tosequences in the T-DNA. The Ti or Ri plasmid also comprises the virregion necessary for the transfer of the T-DNA.

Intermediate vectors cannot replicate themselves in agrobacteria. Theintermediate vector can be transferred into Agrobacterium tumefaciens bymeans of a helper plasmid (conjugation). Binary vectors can replicatethemselves both in E. coli and in agrobacteria. They comprise aselection marker gene and a linker or polylinker which are framed by theright and left T-DNA border regions. They can be transformed directlyinto agrobacteria (Holsters et al., 1978). The agrobacterium used ashost cell is to comprise a plasmid carrying a vir region. The vir regionis necessary for the transfer of the T-DNA into the plant cell.Additional t-DNA may be contained. The bacterium so transformed is usedfor the transformation of plant cells. Plant explants can advantageouslybe cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenesfor the transfer of the DNA into the plant cell. Whole plants can thenbe regenerated from the infected plant material (for example, pieces ofleaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted DNA. No special demands aremade of the plasmids in the case of injection and electroporation. It ispossible to use ordinary plasmids, such as, for example, pUCderivatives. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted. The useof T-DNA for the transformation of plant cells has been intensivelyresearched and sufficiently described in Eur. Pat. Appl. No. EP 120 516;Hockema (1985); An et al., 1985, Herrera-Estrella et al., (1983), Bevanet al., (1983), and Klee et al., (1985).

A particularly useful Ti plasmid cassette vector for transformation ofdicotyledonous plants consists of the enhanced CaMV35S promoter (EN35S)and the 3′ end including polyadenylation signals from a soybean geneencoding the α′-subunit of β-conglycinin. Between these two elements isa multilinker containing multiple restriction sites for the insertion ofgenes of interest.

The vector preferably contains a segment of pBR322 which provides anorigin of replication in E. coli and a region for homologousrecombination with the disarmed T-DNA in Agrobacterium strain ACO; theoriV region from the broad host range plasmid RK1; thestreptomycin/spectinomycin resistance gene from Tn7; and a chimericNPTII gene, containing the CaMV35S promoter and the nopaline synthase(NOS) 3′ end, which provides kanamycin resistance in transformed plantcells.

Optionally, the enhanced CaMV35S promoter may be replaced with the 1.5kb mannopine synthase (MAS) promoter (Velten et al., 1984). Afterincorporation of a DNA construct into the vector, it is introduced intoA. tumefaciens strain ACO which contains a disarmed Ti plasmid.Cointegrate Ti plasmid vectors are selected and subsequentially may beused to transform a dicotyledonous plant.

A. tumefaciens ACO is a disarmed strain similar to pTiB6SE described byFraley et al. (1985). For construction of ACO the starting Agrobacteriumstrain was the strain A208 which contains a nopaline-type Ti plasmid.The Ti plasmid was disarmed in a manner similar to that described byFraley et al. (1985) so that essentially all of the native T-DNA wasremoved except for the left border and a few hundred base pairs of T-DNAinside the left border. The remainder of the T-DNA extending to a pointjust beyond the right border was replaced with a novel piece of DNAincluding (from left to right) a segment of pBR322, the oriV region fromplasmid RK2, and the kanamycin resistance gene from Tn601. The pBR322and oriV segments are similar to these segments and provide a region ofhomology for cointegrate formation.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

4.11.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

4.11.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

4.11.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

Modem Agrobacterium transformation vectors are capable of replication inE. coli as well as Agrobacterium, allowing for convenient manipulationsas described (Klee et al., 1985). Moreover, recent technologicaladvances in vectors for Agrobacterium-mediated gene transfer haveimproved the arrangement of genes and restriction sites in the vectorsto facilitate construction of vectors capable of expressing variouspolypeptide coding genes. The vectors described (Rogers et al., 1987),have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

4.11.4 Gene Expression in Plants

Although great progress has been made in recent years with respect topreparation of transgenic plants which express bacterial proteins suchas B. thuringiensis crystal proteins, the results of expressing nativebacterial genes in plants are often disappointing. Unlike microbialgenetics, little was known by early plant geneticists about the factorswhich affected heterologous expression of foreign genes in plants. Inrecent years, however, several potential factors have been implicated asresponsible in varying degrees for the level of protein expression froma particular coding sequence. For example, scientists now know thatmaintaining a significant level of a particular mRNA in the cell isindeed a critical factor. Unfortunately, the causes for low steady statelevels of mRNA encoding foreign proteins are many. First, full lengthRNA synthesis may not occur at a high frequency. This could, forexample, be caused by the premature termination of RNA duringtranscription or due to unexpected mRNA processing during transcription.Second, full length RNA may be produced in the plant cell, but thenprocessed (splicing, polyA addition) in the nucleus in a fashion thatcreates a nonfunctional mRNA. If the RNA is not properly synthesized,terminated and polyadenylated; it cannot move to the cytoplasm fortranslation. Similarly, in the cytoplasm, if mRNAs have reduced halflives (which are determined by their primary or secondary sequence)insufficient protein product will be produced. In addition, there is aneffect, whose magnitude is uncertain, of translational efficiency onmRNA half-life. In addition, every RNA molecule folds into a particularstructure, or perhaps family of structures, which is determined by itssequence. The particular structure of any RNA might lead to greater orlesser stability in the cytoplasm. Structure per se is probably also adeterminant of mRNA processing in the nucleus. Unfortunately, it isimpossible to predict, and nearly impossible to determine, the structureof any RNA (except for tRNA) in vitro or in vivo. However, it is likelythat dramatically changing the sequence of an RNA will have a largeeffect on its folded structure It is likely that structure per se orparticular structural features also have a role in determining RNAstability.

To overcome these limitations in foreign gene expression, researchershave identified particular sequences and signals in RNAs that have thepotential for having a specific effect on RNA stability. In certainembodiments of the invention, therefore, there is a desire to optimizeexpression of the disclosed nucleic acid segments in planta. Oneparticular method of doing so, is by alteration of the bacterial gene toremove sequences or motifs which decrease expression in a transformedplant cell. The process of engineering a coding sequence for optimalexpression in planta is often referred to as “plantizing” a DNAsequence.

Particularly problematic sequences are those which are A+T rich.Unfortunately, since B. thuringiensis has an A+T rich genome, nativecrystal protein gene sequences must often be modified for optimalexpression in a plant. The sequence motif ATTTA (or AUUUA as it appearsin RNA) has been implicated as a destabilizing sequence in mammaliancell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich3′ untranslated regions, and these regions often have the ATTTAsequence, sometimes present in multiple copies or as multimers (e.g.,ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ endof an unstable mRNA to a stable RNA (globin or VA1) decreased the stableRNA's half life dramatically. They further showed that a pentamer ofATTTA had a profound destabilizing effect on a stable message, and thatthis signal could exert its effect whether it was located at the 3′ endor within the coding sequence. However, the number of ATTTA sequencesand/or the sequence context in which they occur also appear to beimportant in determining whether they function as destabilizingsequences. Shaw and Kamen showed that a trimer of ATTTA had much lesseffect than a pentamer on mRNA stability and a dimer or a monomer had noeffect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTAsuch as a pentamer automatically create an A+T rich region. This wasshown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs,the ATTTA sequence may be present in only a single copy, but it is oftencontained in an A+T rich region. From the animal cell data collected todate, it appears that ATTTA at least in some contexts is important instability, but it is not yet possible to predict which occurrences ofATTTA are destabiling elements or whether any of these effects arelikely to be seen in plants.

Some studies on mRNA degradation in animal cells also indicate that RNAdegradation may begin in some cases with nucleolytic attack in A+T richregions. It is not clear if these cleavages occur at ATTTA sequences.There are also examples of mRNAs that have differential stabilitydepending on the cell type in which they are expressed or on the stagewithin the cell cycle at which they are expressed. For example, histonemRNAs are stable during DNA synthesis but unstable if DNA synthesis isdisrupted. The 3′ end of some histone mRNAs seems to be responsible forthis effect (Pandey and Marzluff, 1987). It does not appear to bemediated by ATTTA, nor is it clear what controls the differentialstability of this mRNA. Another example is the differential stability ofIgG mRNA in B lymphocytes during B cell maturation (Genovese andMilcarek, 1988). A final example is the instability of a mutantβ-thallesemic globin mRNA. In bone marrow cells, where this gene isnormally expressed, the mutant mRNA is unstable, while the wild-typemRNA is stable. When the mutant gene is expressed in HeLa or L cells invitro, the mutant mRNA shows no instability (Lim et al., 1988). Theseexamples all provide evidence that mRNA stability can be mediated bycell type or cell cycle specific factors. Furthermore this type ofinstability is not yet associated with specific sequences. Given theseuncertainties, it is not possible to predict which RNAs are likely to beunstable in a given cell. In addition, even the ATTTA motif may actdifferentially depending on the nature of the cell in which the RNA ispresent. Shaw and Kamen (1987) have reported that activation of proteinkinase C can block degradation mediated by ATTTA.

The addition of a polyadenylate string to the 3′ end is common to mosteukaryotic mRNAs, both plant and animal. The currently accepted view ofpolyA addition is that the nascent transcript extends beyond the mature3′ terminus. Contained within this transcript are signals forpolyadenylation and proper 3′ end formation. This processing at the 3′end involves cleavage of the mRNA and addition of polyA to the mature 3′end. By searching for consensus sequences near the polyA tract in bothplant and animal mRNAs, it has been possible to identify consensussequences that apparently are involved in polyA addition and 3′ endcleavage. The same consensus sequences seem to be important to both ofthese processes. These signals are typically a variation on the sequenceAATAAA. In animal cells, some variants of this sequence that arefunctional have been identified; in plant cells there seems to be anextended range of functional sequences (Wickens and Stephenson, 1984;Dean et al., 1986). Because all of these consensus sequences arevariations on AATAAA, they all are A+T rich sequences. This sequence istypically found 15 to 20 bp before the polyA tract in a mature mRNA.Studies in animal cells indicate that this sequence is involved in bothpolyA addition and 3′ maturation. Site directed mutations in thissequence can disrupt these functions (Conway and Wickens, 1988; Wickenset al., 1987). However, it has also been observed that sequences up to50 to 100 bp 3′ to the putative polyA signal are also required; i.e., agene that has a normal AATAAA but has been replaced or disrupteddownstream does not get properly polyadenylated (Gil and Proudfoot,1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, thepolyA signal itself is not sufficient for complete and properprocessing. It is not yet known what specific downstream sequences arerequired in addition to the polyA signal, or if there is a specificsequence that has this function. Therefore, sequence analysis can onlyidentify potential polyA signals.

In naturally occurring mRNAs that are normally polyadenylated, it hasbeen observed that disruption of this process, either by altering thepolyA signal or other sequences in the mRNA, profound effects can beobtained in the level of functional mRNA. This has been observed inseveral naturally occurring mRNAs, with results that are gene-specificso far.

It has been shown that in natural mRNAs proper polyadenylation isimportant in mRNA accumulation, and that disruption of this process caneffect mRNA levels significantly. However, insufficient knowledge existsto predict the effect of changes in a normal gene. In a heterologousgene, it is even harder to predict the consequences. However, it ispossible that the putative sites identified are dysfunctional. That is,these sites may not act as proper polyA sites, but instead function asaberrant sites that give rise to unstable mRNAs.

In animal cell systems, AATAAA is by far the most common signalidentified in mRNAs upstream of the polyA, but at least four variantshave also been found (Wickens and Stephenson, 1984). In plants, notnearly so much analysis has been done, but it is clear that multiplesequences similar to AATAAA can be used. The plant sites in Table 5called major or minor refer only to the study of Dean et al. (1986)which analyzed only three types of plant gene. The designation ofpolyadenylation sites as major or minor refers only to the frequency oftheir occurrence as functional sites in naturally occurring genes thathave been analyzed. In the case of plants this is a very limiteddatabase. It is hard to predict with any certainty that a sitedesignated major or minor is more or less likely to function partiallyor completely when found in a heterologous gene such as those encodingthe crystal proteins of the present invention.

TABLE 5 POLYADENYLATION SITES IN PLANT GENES PA AATAAA Major consensussite P1A AATAAT Major plant site P2A AACCAA Minor plant site P3A ATATAA″ P4A AATCAA ″ P5A ATACTA ″ P6A ATAAAA ″ P7A ATGAAA ″ P8A AAGCAT ″ P9AATTAAT ″ P10A ATACAT ″ P11A AAAATA ″ P12A ATTAAA Minor animal site P13AAATTAA ″ P14A AATACA ″ P15A CATAAA ″

The present invention provides a method for preparing synthetic plantgenes which genes express their protein product at levels significantlyhigher than the wild-type genes which were commonly employed in planttransformation heretofore. In another aspect, the present invention alsoprovides novel synthetic plant genes which encode non-plant proteins.

As described above, the expression of native B. thuringiensis genes inplants is often problematic. The nature of the coding sequences of B.thuringiensis genes distinguishes them from plant genes as well as manyother heterologous genes expressed in plants. In particular, B.thuringiensis genes are very rich (˜62%) in adenine (A) and thymine (T)while plant genes and most other bacterial genes which have beenexpressed in plants are on the order of 45-55% A+T.

Due to the degeneracy of the genetic code and the limited number ofcodon choices for any amino acid, most of the “excess” A+T of thestructural coding sequences of some Bacillus species are found in thethird position of the codons. That is, genes of some Bacillus specieshave A or T as the third nucleotide in many codons. Thus A+T content inpart can determine codon usage bias. In addition, it is clear that genesevolve for maximum function in the organism in which they evolve. Thismeans that particular nucleotide sequences found in a gene from oneorganism, where they may play no role except to code for a particularstretch of amino acids, have the potential to be recognized as genecontrol elements in another organism (such as transcriptional promotersor terminators, polyA addition sites, intron splice sites, or specificmRNA degradation signals). It is perhaps surprising that such misreadsignals are not a more common feature of heterologous gene expression,but this can be explained in part by the relatively homogeneous A+Tcontent (˜50%) of many organisms. This A+T content plus the nature ofthe genetic code put clear constraints on the likelihood of occurrenceof any particular oligonucleotide sequence. Thus, a gene from E. coliwith a 50% A+T content is much less likely to contain any particular A+Trich segment than a gene from B. thuringiensis.

Typically, to obtain high-level expression of the S-endotoxin genes inplants, existing structural coding sequence (“structural gene”) whichcodes for the S-endotoxin are modified by removal of ATTTA sequences andputative polyadenylation signals by site directed mutagenesis of the DNAcomprising the structural gene. It is most preferred that substantiallyall the polyadenylation signals and ATTTA sequences are removed althoughenhanced expression levels are observed with only partial removal ofeither of the above identified sequences. Alternately if a syntheticgene is prepared which codes for the expression of the subject protein,codons are selected to avoid the ATTTA sequence and putativepolyadenylation signals. For purposes of the present invention putativepolyadenylation signals include, but are not necessarily limited to,AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT,ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. In replacingthe ATTTA sequences and polyadenylation signals, codons are preferablyutilized which avoid the codons which are rarely found in plant genomes.

The selected DNA sequence is scanned to identify regions with greaterthan four consecutive adenine (A) or thymine (T) nucleotides. The A+Tregions are scanned for potential plant polyadenylation signals.Although the absence of five or more consecutive A or T nucleotideseliminates most plant polyadenylation signals, if there are more thanone of the minor polyadenylation signals identified within tennucleotides of each other, then the nucleotide sequence of this regionis preferably altered to remove these signals while maintaining theoriginal encoded amino acid sequence.

The second step is to consider the about 15 to about 30 or so nucleotideresidues surrounding the A+T rich region identified in step one. If theA+T content of the surrounding region is less than 80%, the regionshould be examined for polyadenylation signals. Alteration of the regionbased on polyadenylation signals is dependent upon (1) the number ofpolyadenylation signals present and (2) presence of a major plantpolyadenylation signal.

The extended region is examined for the presence of plantpolyadenylation signals. The polyadenylation signals are removed bysite-directed mutagenesis of the DNA sequence. The extended region isalso examined for multiple copies of the ATTTA sequence which are alsoremoved by mutagenesis.

It is also preferred that regions comprising many consecutive A+T basesor G+C bases are disrupted since these regions are predicted to have ahigher likelihood to form hairpin structure due to self-complementarity.Therefore, insertion of heterogeneous base pairs would reduce thelikelihood of self-complementary secondary structure formation which areknown to inhibit transcription and/or translation in some organisms. Inmost cases, the adverse effects may be minimized by using sequenceswhich do not contain more than five consecutive A+T or G+C.

4.11.5 Synthetic Oligonucleotides for Mutagenesis

When oligonucleotides are used in the mutagenesis, it is desirable tomaintain the proper amino acid sequence and reading frame, withoutintroducing common restriction sites such as BglII, HindIII, SacI, KpnI,EcoRI, NcoI, PstI and SalI into the modified gene. These restrictionsites are found in poly-linker insertion sites of many cloning vectors.Of course, the introduction of new polyadenylation signals, ATTTAsequences or consecutive stretches of more than five A+T or G+C, shouldalso be avoided. The preferred size for the oligonucleotides is about 40to about 50 bases, but fragments ranging from about 18 to about 100bases have been utilized. In most cases, a minimum of about 5 to about 8base pairs of homology to the template DNA on both ends of thesynthesized fragment are maintained to insure proper hybridization ofthe primer to the template. The oligonucleotides should avoid sequenceslonger than five base pairs A+T or G+C. Codons used in the replacementof wild-type codons should preferably avoid the TA or CG doubletwherever possible. Codons are selected from a plant preferred codontable (such as Table 6 below) so as to avoid codons which are rarelyfound in plant genomes, and efforts should be made to select codons topreferably adjust the G+C content to about 50%.

TABLE 6 PREFERRED CODON USAGE IN PLANTS Percent Usage Amino Acid Codonin Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29 AGG 23 LEU CUA 8 CUC 20CUG 10 CUU 28 UUA 5 UUG 30 SER UCA 14 UCC 26 UCG 3 UCU 21 AGC 21 AGU 15THR ACA 21 ACC 41 ACG 7 ACU 31 PRO CCA 45 CCC 19 CCG 9 CCU 26 ALA GCA 23GCC 32 GCG 3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45AUU 43 VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU28 GLN CAA 64 CAG 36 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU52 TYR UAC 68 UAU 32 CYS UGC 78 UGU 22 PHE UUC 56 UUU 44 MET AUG 100 TRPUGG 100

Regions with many consecutive A+T bases or G+C bases are predicted tohave a higher likelihood to form hairpin structures due toself-complementarity. Disruption of these regions by the insertion ofheterogeneous base pairs is preferred and should reduce the likelihoodof the formation of self-complementary secondary structures such ashairpins which are known in some organisms to inhibit transcription(transcriptional terminators) and translation (attenuators).

Alternatively, a completely synthetic gene for a given amino acidsequence can be prepared, with regions of five or more consecutive A+Tor G+C nucleotides being avoided. Codons are selected avoiding the TAand CG doublets in codons whenever possible. Codon usage can benormalized against a plant preferred codon usage table (such as Table 6)and the G+C content preferably adjusted to about 50%. The resultingsequence should be examined to ensure that there are minimal putativeplant polyadenylation signals and ATTTA sequences. Restriction sitesfound in commonly used cloning vectors are also preferably avoided.However, placement of several unique restriction sites throughout thegene is useful for analysis of gene expression or construction of genevariants.

4.11.6 “Plantized” Gene Constructs

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA. Transcription of DNA into mRNA is regulated by a region ofDNA usually referred to as the “promoter.” The promoter region containsa sequence of bases that signals RNA polymerase to associate with theDNA and to initiate the transcription of mRNA using one of the DNAstrands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the CauliflowerMosaic Virus (CaMV) 19S and 35S promoters, the light-inducible promoterfrom the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO,a very abundant plant polypeptide) and the mannopine synthase (MAS)promoter (Velten et al., 1984 and Velten and Schell, 1985). All of thesepromoters have been used to create various types of DNA constructs whichhave been expressed in plants (see e.g, Int. Pat. Appl. Publ. No. WO84/02913).

Promoters which are known or are found to cause transcription of RNA inplant cells can be used in the present invention. Such promoters may beobtained from plants or plant viruses and include, but are not limitedto, the CaMV35S promoter and promoters isolated from plant genes such asssRUBISCO genes. As described below, it is preferred that the particularpromoter selected should be capable of causing sufficient expression toresult in the production of an effective amount of protein.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in thefollowing examples. Rather, the non-translated leader sequence can bepart of the 5′ end of the non-translated region of the coding sequencefor the virus coat protein, or part of the promoter sequence, or can bederived from an unrelated promoter or coding sequence. In any case, itis preferred that the sequence flanking the initiation site conform tothe translational consensus sequence rules for enhanced translationinitiation reported by Kozak (1984).

The cry DNA constructs of the present invention may also contain one ormore modified or fully-synthetic structural coding sequences which havebeen changed to enhance the performance of the cry gene in plants. Thestructural genes of the present invention may optionally encode a fusionprotein comprising an amino-terminal chloroplast transit peptide orsecretory signal sequence.

The DNA construct also contains a 3′ non-translated region. The 3′non-translated region contains a polyadenylation signal which functionsin plants to cause the addition of polyadenylate nucleotides to the 3′end of the viral RNA. Examples of suitable 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylationsignal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as thenopaline synthase (NOS) gene, and (2) plant genes like the soybeanstorage protein (7S) genes and the small subunit of the RuBP carboxylase(E9) gene.

4.12 Methods for Producting Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with arecombinant cry* gene-containing segment, the expression of the encodedcrystal protein (i.e., a bacterial crystal protein or polypeptide havinginsecticidal activity against coleopterans) can result in the formationof insect-resistant plants.

By way of example, one may utilize an expression vector containing acoding region for a B. thuringiensis crystal protein and an appropriateselectable marker to transform a suspension of embryonic plant cells,such as wheat or corn cells using a method such as particle bombardment(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated onmicroprojectiles into the recipient cells. Transgenic plants are thenregenerated from transformed embryonic calli that express theinsecticidal proteins.

The formation of transgenic plants may also be accomplished using othermethods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, 1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

Such plants can form germ cells and transmit the transformed trait(s) toprogeny plants. Likewise, transgenic plants can be grown in the normalmanner and crossed with plants that have the same transformed hereditaryfactors or other hereditary factors. The resulting hybrid individualshave the corresponding phenotypic properties. A transgenic plant of thisinvention thus has an increased amount of a coding region (e.g., amutated cry gene) that encodes the mutated Cry polypeptide of interest.A preferred transgenic plant is an independent segregant and cantransmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating.

Seed from a transgenic plant may be grown in the field or greenhouse,and resulting sexually mature transgenic plants are self-pollinated togenerate true breeding plants. The progeny from these plants become truebreeding lines that are evaluated for, by way of example, increasedinsecticidal capacity against coleopteran insects, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic plants of commercial interest includingvarious grasses, grains, fibers, tubers, legumes, ornamental plants,cacti, succulents, fruits, berries, and vegetables, as well as a numberof nut- and fruit-bearing trees and plants.

4.13 Methods for Producing Combinatorial Cry3* Variants

Crystal protein mutants containing substitutions in one or more domainsmay be constructed via a number of techniques. For instance, sequencesof highly related genes can be readily shuffled using the PCR™-basedtechnique described by Stemmer (1994). Alternatively, if suitablerestriction sites are available, the mutations of one cry gene may becombined with the mutations of a second cry gene by routine subcloningmethodologies. If a suitable restriction site is not available, one maybe generated by oligonucleotide directed mutagenesis using any number ofprocedures known to those skilled in the art. Alternatively,splice-overlap extension PCR™ (Horton et al., 1989) may be used tocombine mutations in different regions of a crystal protein. In thisprocedure, overlapping DNA fragments generated by the PCR™ andcontaining different mutations within their unique sequences may beannealed and used as a template for amplification using flanking primersto generate a hybrid gene sequence. Finally, cry* mutants may becombined by simply using one cry mutant as a template foroligonucleotide-directed mutagenesis using any number of protocols suchas those described herein.

4.14 Isolating Homologous Gene and Gene Fragments

The genes and δ-endotoxins according to the subject invention includenot only the full length sequences disclosed herein but also fragmentsof these sequences, or fusion proteins, which retain the characteristicinsecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person skill in this art that insecticidalδ-endotoxins can be identified and obtained through several means. Thespecific genes, or portions thereof, may be obtained from a culturedepository, or constructed synthetically, for example, by use of a genemachine. Variations of these genes may be readily constructed usingstandard techniques for making point mutations. Also, fragments of thesegenes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes which codefor active fragments may be obtained using a variety of otherrestriction enzymes. Proteases may be used to directly obtain activefragments of these δ-endotoxins.

Equivalent δ-endotoxins and/or genes encoding these equivalentδ-endotoxins can also be isolated from Bacillus strains and/or DNAlibraries using the teachings provided herein. For example, antibodiesto the δ-endotoxins disclosed and claimed herein can be used to identifyand isolate other δ-endotoxins from a mixture of proteins. Specifically,antibodies may be raised to the portions of the δ-endotoxins which aremost constant and most distinct from other B. thuringiensisδ-endotoxins. These antibodies can then be used to specifically identifyequivalent δ-endotoxins with the characteristic insecticidal activity byimmunoprecipitation, enzyme linked immunoassay (ELISA), or Westernblotting.

A further method for identifying the δ-endotoxins and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are nucleotide sequences having a detectable label. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and sample are essentially identical.The probe's detectable label provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying formicidal δ-endotoxin genes ofthe subject invention.

The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin orthyroxine, as well as enzymes such as hydrolases or peroxidases, or thevarious chemiluminescers such as luciferin, or fluorescent compoundslike fluorescein and its derivatives. The probe may also be labeled atboth ends with different types of labels for ease of separation, as, forexample, by using an isotopic label at the end mentioned above and abiotin label at the other end.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probes of thesubject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

The potential variations in the probes listed is due, in part, to theredundancy of the genetic code. Because of the redundancy of the geneticcode, i.e., more than one coding nucleotide triplet (codon) can be usedfor most of the amino acids used to make proteins. Therefore differentnucleotide sequences can code for a particular amino acid. Thus, theamino acid sequences of the B. thuringiensis δ-endotoxins and peptidescan be prepared by equivalent nucleotide sequences encoding the sameamino acid sequence of the protein or peptide. Accordingly, the subjectinvention includes such equivalent nucleotide sequences. Also, inverseor complement sequences are an aspect of the subject invention and canbe readily used by a person skilled in this art. In addition it has beenshown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser and Kezdy, 1984). Thus,the subject invention includes mutants of the amino acid sequencedepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is substantiallyretained. Further, the invention also includes mutants of organismshosting all or part of a δ-endotoxin encoding a gene of the invention.Such mutants can be made by techniques well known to persons skilled inthe art. For example, UV irradiation can be used to prepare mutants ofhost organisms. Likewise, such mutants may include asporogenous hostcells which also can be prepared by procedures well known in the art.

4.15 Ribozymes

Ribozymes are enzymatic RNA molecules which cleave particular mRNAspecies. In certain embodiments, the inventors contemplate the selectionand utilization of ribozymes capable of cleaving the RNA segments of thepresent invention, and their use to reduce activity of target mRNAs inparticular cell types or tissues.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992);examples of hairpin motifs are described by Hampel et al. (Eur. Pat. EP0360257), Hampel and Tritz (1989), Hampel et al. (1990) and Cech et al.(U.S. Pat. No. 5,631,359; an example of the hepatitis δ virus motif isdescribed by Perrotta and Been (1992); an example of the RNaseP motif isdescribed by Guerrier-Takada et al. (1983); Neurospora VS RNA ribozymemotif is described by Collins (Saville and Collins, 1990; Saville andCollins, 1991; Collins and Olive, 1993); and an example of the Group Iintron is described by Cech et al. (U.S. Pat. No. 4,987,071). All thatis important in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule. Thus the ribozyme constructsneed not be limited to specific motifs mentioned herein.

The invention provides a method for producing a class of enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target. The enzymatic nucleic acid molecule is preferablytargeted to a highly conserved sequence region of a target mRNA suchthat specific treatment of a disease or condition can be provided witheither one or several enzymatic nucleic acids. Such enzymatic nucleicacid molecules can be delivered exogenously to specific cells asrequired. Alternatively, the ribozymes can be expressed from DNA or RNAvectors that are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Draper et al., Int. Pat. Appl. Publ. No. WO 93/23569, andSullivan et al., Int. Pat. Appl. Publ. No. WO 94/02595, both herebyincorporated in their totality by reference herein; Ohkawa et al, 1992;Taira et al., 1991; Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Draper et al. (Int. Pat. Appl.Publ. No. WO 93/23569), or Sullivan et al., (Int. Pat. Appl. Publ. No.WO 94/02595) and synthesized to be tested in vitro and in vivo, asdescribed. Such ribozymes can also be optimized for delivery. Whilespecific examples are provided, those in the art will recognize thatequivalent RNA targets in other species can be utilized when necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 baseson each arm are able to bind to, or otherwise interact with, the targetRNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H(for a review see Usman and Cedergren, 1992). Ribozymes may be purifiedby gel electrophoresis using general methods or by high pressure liquidchromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) andDraper et al. (Int. Pat. Appl. Publ. No. WO 93/23569) which have beenincorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells (e.gKashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations within cell lines or cell types. They canalso be used to assess levels of the target RNA molecule. The closerelationship between ribozyme activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple ribozymes described in this invention, onemay map nucleotide changes which are important to RNA structure andfunction in vitro, as well as in cells and tissues. Cleavage of targetRNAs with ribozymes may be used to inhibit gene expression and definethe role (essentially) of specified gene products in particular cells orcell types.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1 Three-Dimensional Structure of Cry3Bb

The three-dimensional structure of Cry3Bb was determined by X-raycrystallography. Crystallization of Cry3Bb and X-ray diffraction datacollection were performed as described by Cody et al. (1992). Thecrystal structure of Cry3Bb was refined to a residual R factor of 18.0%using data collected to 2.4 Å resolution. The crystals belong to thespace group C222₁ with unit cell dimensions a=122.44, b=131.81, andc=105.37 Å and contain one molecule in the asymmetric unit. Atomiccoordinates for Cry3Bb are described in Example 31 and listed in Section9.

The structure of Cry3Bb is similar to that of Cry3A (Li et al., 1991).It consists of 5825 protein atoms from 588 residues (amino acids 64-652)forming three discrete domains (FIG. 1). A total of 251 water moleculeshave been identified in the Cry3Bb structure (FIG. 2). Domain 1(residues 64-294) is a seven helical bundle formed by six helicestwisted around the central helix, α5 (FIG. 3). The amino acids formingeach helix are listed in FIG. 4. Domain 2 (residues 295-502) containsthree antiparallel β-sheets (FIG. 5A and FIG. 5B). Sheets 1 and 2, eachcomposed of 4 β strands, form the distinctive “Greek key” motif. Theouter surface of sheet 3, composed of 3 β strands, makes contact withhelix α7 of domain 1. FIG. 6 lists the amino acids comprising each βstrand in domain 2. A small α helix, α8 which follows β strand 1, isalso included in domain 2. Domain 3 (residues 503-652) has a “jellyroll” β-barrel topology which has a hydrophobic core and is nearlyparallel to the a and perpendicular to the c axes of the lattice (FIG.7A and FIG. 7B). The amino acids comprising each β strand of domain 3are listed in FIG. 8.

The monomers of Cry3Bb in the crystal form a dimeric quaternarystructure along a two-fold axis parallel to the a axis (FIG. 9A and FIG.9B). Helix α6 lies in a cleft formed by the interface of domain 1 anddomains 1 and 3 of its symmetry related molecule. There are numerousclose hydrogen bonding contacts along this surface, confirming thestructural stability of the dimer.

5.2 Example 2 Preparation of Cry3Bb.60

B. thuringiensis EG7231 was grown through sporulation in C2 medium withchloramphenicol (Cml) selection. The solids from this culture wererecovered by centrifugation and washed with water. The toxin waspurified by recrystallization from 4.0 M NaBr (Cody et al., 1992). Thepurified Cry3Bb was solubilized in 10 ml of 50 mM KOH/100 mg Cry3Bb andbuffered to pH 9.0 with 100 mM CAPS (pH 9.0). The soluble toxin wastreated with trypsin at a weight ratio of 50 mg toxin to 1 mg trypsin.After 20 min of trypsin digestion the predominant protein visualized bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) was 60 kDa. Furtherdigestion of the 60-kDa toxin was not observed. FIG. 4 illustrates theCoomassie-stained Cry3Bb and Cry3Bb.60 following SDS-PAGE.

5.3 Example 3 Purification and Sequencing of Cry3Bb.60

Cry3Bb.60 was electrophoretically purified by SDS-PAGE andelectroblotted to Immobilon-P® (Millipore) membrane by semi-dry transferat 15V for 30 min. The membrane was then washed twice with water andstained with 0.025% R-250, 40% methanol. To reduce the background, theblot was destained with 50% methanol until the stained protein bandswere visible. The blot was then air dried, and the stained Cry3Bb.60band was cut out of the membrane. This band was sent to the TuftsUniversity Sequencing Laboratory (Boston, Mass.) for N-terminalsequencing. The experimentally-determined N-terminal amino acid sequenceis shown in Table 7 beside the known amino acid sequence starting atamino acid residue 160.

TABLE 7 AMINO ACID SEQUENCE OF THE N-TERMINUS OF CRY3BB.60 ANDCOMPARISON TO THE KNOWN SEQUENCE OF CRY3BB Deduced Known SequenceSequence Residue # S S 160 K K 161 R R 162 S S 163 Q Q 164 D D 165 R R166

5.4 Example 4 Bioactivity of Cry3Bb.60

Cry3Bb was prepared for bioassay by solubilization in a minimal amountof 50 mM KOH, 10 ml per 100 mg toxin, and buffered to pH 9.0 with 100 mMCAPS, pH 9.0. Cry3Bb.60 was prepared as described in Example 1. Bothpreparations were kept at room temperature 12 to 16 hours prior tobioassay. After seven days the mortality of the population wasdetermined and analyzed to determine the lethal concentration of eachtoxin. These results are numerized in Table 8.

TABLE 8 BIOACTIVITY OF CRY3BB AND CRY3BB.60 AGAINST THE SOUTHERN CORNROOTWORM (DIABIOTICA UNDECIMPUNCTATA) LC₅₀ mg/well 95% C. I. Cry3Bb24.09 15–39 Cry3Bb.60  6.72 5.25–8.4 

5.5 Example 5 Ion-Channel Formation by Cry3Bb and CryB2.60

Cry3Bb.60 and Cry3Bb were evaluated for their ability to form ionchannels in planar lipid bilayers. Bilayers of phosphatidylcholine wereformed on Teflon® supports over a 0.7-mm hole. A bathing solution of 3.5ml 100 mM KOH, 10 mM CaCl₂, 100 mM CAPS (pH 9.5) was placed on eitherside of the Teflon® partition. The toxin was added to one side of thepartition and a voltage of 60 mV was imposed across thephosphatidylcholine bilayer. Any leakage of ions through the membranewas amplified and recorded. An analysis of the frequency of theconductances created by either Cry3Bb or Cry3Bb.60 are illustrated inFIG. 5A and FIG. 5B. Cry3Bb.60 readily formed ion channels whereasCry3Bb rarely formed channels.

5.6 Example 6 Formation of High Molecular-Weight Oligomers

Individual molecules of Cry3Bb or Cry3Bb.60 form a complex with anotherlike molecule. The ability of Cry3Bb to form an oligomer is notreproducibly apparent. The complex cannot be repeatedly observed to formunder nondenaturing conditions. Cry3Bb.60 formed a significantly greateramount of a higher molecular-weight complex (≧120 kDa) with otherCry3Bb.60 molecules. Oligomers of Cry3Bb are demonstrated by theintensity of the Coomassie-stained SDS polyacrylamide gel.Oligomerization is visualized on SDS-PAGE by not heating samples priorto loading on the gel to retain some nondenatured toxin. These datasuggest that Cry3Bb.60 more readily forms the higher order complex thanCry3Bb alone. Oligomerization is also observed by studying theconductance produced by these molecules and the time-dependent increasein conductance. This change in conductance can be attributed tooligomerization of the toxin.

5.7 Example 7 Design Method 1: Identification and Alteration ofProtease-Sensitive Sites and Proteolytic Processing

It has been reported in the literature that treatment of Cry3A toxinprotein with trypsin, an enzyme that cleaves proteins on the carboxylside of available lysine and arginine residues, yields a stable cleavageproduct of 55 kDa from the 67 kDa native protein (Carroll et al., 1989).N-terminal sequencing of the 55 kDa product showed cleavage occurs atamino acid residue R158. The truncated Cry3A protein was found to retainthe same level of insecticidal activity as the native protein. Cry3Bbtoxin protein was also treated with trypsin. After digestion, theprotein size decreased from 68 kDa, the molecular weight of the nativeCry3Bb toxin, to 60 kDa. No further digestion was observed. N-terminalsequencing revealed the trypsin cleavage site of the truncated toxin(Cry3Bb.60) to be amino acid R159 in lα3,4 of Cry3Bb. Unexpectedly, thebioactivity of the truncated Cry3Bb toxin was found to increase.

Using this method, protease digestion of a B. thuringiensis toxinprotein, a proteolytically sensitive site was identified on Cry3Bb, anda more highly active form of the protein (Cry3Bb.60) was identified.Modifications to this proteolytically-sensitive site by introducing anadditional protease recognition site also resulted in the isolation of abiologically more active protein. It is also possible that removal ofother protease-sensitive site(s) may improve activity. Proteolyticallysensitive regions, once identified, may be modified or utilized toproduce biologically more active toxins.

5.7.1 Cry3Bb.60

Treatment of solubilized Cry3Bb toxin protein with trypsin results inthe isolation of a stable, truncated Cry3Bb toxin protein with amolecular weight of 60 kDa (Cry3Bb.60). N-terminal sequencing ofCry3Bb.60 shows the trypsin-sensitive site to be R159 in lα3,4 of thenative toxin. Trypsin digestion results in the removal of helices 1-3from the native Cry3Bb but also increases the activity of the toxinagainst SCRW larvae approximately four-fold.

Cry3Bb.60 is a unique toxin with enhanced insecticidal use over theparent Cry3Bb. Improved biological activity, is only one parameter thatdistinguishes it as a new toxin. Aside from the reduced size, Cry3Bb.60is also a more soluble protein. Cry3Bb precipitates from solution at pH6.5 while Cry3Bb.60 remains in solution from pH 4.5 to pH 12. Cry3Bb.60also forms ion channels with greater frequency than Cry3Bb.

Cry3Bb.60 is produced by either the proteolytic removal of the first 159amino acid residues, or the in vivo production of this toxin, bybacteria or plants expressing the gene for Cry3Bb.60, that is, theCry3Bb gene without the first 483 nucleotides.

In conclusion, Cry3Bb.60 is distinct from Cry3Bb in several importantways: enhanced insecticidal activity; enhanced range of solubility;enhanced ability to form channels; and reduced size.

5.7.2 EG11221

Semi-random mutagenesis of the trypsin-sensitive lα3,4 region of Cry3Bbresulted in the isolation of Cry3Bb.11221, a designed Cry3Bb proteinthat exhibits over a 6-fold increase in activity against SCRW larvaecompared to WT. Cry3Bb.11221 has 4 amino acid changes in the lα3,4region. One of these changes, L158R, introduces an additional trypsinsite adjacent to R159, the proteolytically sensitive site used toproduce Cry3Bb.60 (example 4.1.1). Cry3Bb.11221 is produced by B.thuringiensis as a full length toxin protein but is presumably digestedby insect gut proteases to the same size as Cry3Bb.60 (see Cry3A resultsfrom Carroll et al., 1989). The additional protease recognition site maymake the lα3,4 region even more sensitive to digestion, therebyincreasing activity.

5.8 Example 8 Design Method 2: Determining and Manipulation of BoundWater

There are several ways that water molecules can associate with aprotein, including surface water that is easily removed and bound waterthat is more difficult to extract (Dunitz, 1994; Zhang and Matthews,1994). The function of bound water has been the subject of significantacademic extrapolation, but the precise function has little experimentalvalidation. Some of the most interesting bound or structural water isthe water that participates in the protein structure from inside theprotein itself.

The occupation of a site by a water molecule can indicate a stablepocket within a protein or a looseness of packing created bywater-mediated salt bridges and hydrogen bonding to water. This canreduce the degree of bonding between amino acids, possibly making theregion more flexible. A different amino acid sequence around that samesite could result in better packing, collapsing the pocket around polaror charged amino acids. This may result in decreased flexibility.Therefore, the degree of hydration of a region of a protein maydetermine the flexibility or mobility of that region, and manipulationof the hydration may alter the flexibility. Methods of increasing thehydration of a water-exposed region include increasing the number ofhydrophobic residues along that surface. It is taught in the art thatexposed hydrophobic residues require significantly more water to hydratethan hydrophilic residues (CRC Handbook of Chemistry and Physics, CRCPress, Inc.). It is not taught, however, that by doing this,improvements to the biological activity of a protein can be achieved.

Structural water has not previously been identified in B. thuringiensisδ-endotoxins including Cry3Bb. Furthermore, there are no reports of thefunction of this structural water in δ-endotoxins or bacterial toxins.In the analysis of Cry3Bb, it was observed that a collection of watermolecules are located around lα3,4, a site defined by the inventors asimportant for improvement of bioactivity. The loop α3,4 region issurface exposed and may define a hinge in the protein permitting eitherremoval or movement of the first three helices of domain 1. Thehydration found around this region may impart flexibility and mobilityto this loop. The observation of structural water at the lα3,4 siteprovided an analytical tool for further structure analysis. If thisimportant site is surrounded by water, then other important sites mayalso be completely or partially surrounded by water. Using this insight,structural water surrounding helices 5 and 6 was then identified. Thisstructural water forms a column through the protein, effectivelyseparating helices 5 and 6 from the rest of the molecule. The structuresof Cry3A and Cry3Bb suggest that helices 5 and 6 are tightly associated,bound together by Van der Waals interactions. Alone, helix 5 from Cry3A,although insufficient for biological activity, has been demonstrated tohave the ability to form ion channels in an artificial membrane (Gazitand Shai, 1993). The ion channels formed by helix 5 are 10-fold smallerthan the channels of the full length toxin suggesting that significantlymore toxin structure is required for the full-sized ion channels. InCry3Bb, helix 5 as part of a cluster of α helices (domain 1) has beenfound to form ion channels (Von Tersch et al., 1994). Unpublishedexperimental observations by the inventors demonstrate that helix 6 alsocrossed the biological membrane. Helices 5 and 6, therefore, are theputative channel-forming helices necessary for toxicity.

The hydration around these helices may indicate that flexibility of thisregion is necessary for toxicity. It is conceivable, therefore, that ifit were possible to improve the hydration around helices 5 and 6, onecould create a better toxin protein. Care must be taken, however, toavoid creating continuous hydrophobic surfaces between helices 5-6 andany other part of the protein which could, by hydrophobic interactions,act to restrict movement of the mobile helices. The mobility of helices5 and 6 may also depend on the flexibility of the loops attached to themas well as on other regions of the Cry3Bb molecule, particularly indomain 1, which may undergo conformational changes to allow insertion ofthe 2 helices into the membrane. Altering the hydration of these regionsof the protein may also affect its bioactivity.

5.8.1 Cry3Bb.11032

A collection of bound water residues indicated the relative flexibilityof the lα3,4 region. The flexibility of this loop can be increased byincreasing the hydration of the region by substituting relativelyhydrophobic residues for the exposed hydrophilic residues. An example ofan improved, designed protein having this type of substitution isCry3Bb.11032. Cry3Bb.11032 has the amino acid change D165G; glycine ismore hydrophobic than aspartate (Kyte and Doolittle hydrophobicity scoreof −0.4 vs. −3.5 for aspartate). Cry3Bb.11032 is approximately 3 timesmore active than WT Cry3Bb.

5.8.2 Cry3Bb.11051

To increase the hydration of the lα4,5 region of Cry3Bb, glycine wassubstituted for the surface exposed residue K189. Glycine is morehydrophobic than lysine (Kyte and Doolittle hydrophobicity score of −0.4vs. −3.9 for lysine) and may result in an increase in bound water. Theincrease in bound water may impart greater flexibility to the loopregion which precedes the channel-forming helix, α5. The designed Cry3Bbprotein with the K189G change, Cry3Bb.11051, exhibits a 3-fold increasein activity compared to WT Cry3Bb.

5.8.3 Alterations to Lα7,β1 (Cry3Bb.11241 and 11242)

Amino acid changes made in the surface-exposed loop connecting α-helix 7and β-strand 1 (lα7,β1) resulted in the identification of 2 alteredCry3Bb proteins with increased bioactivities, Cry3Bb.11241 andCry3Bb.11242. Analysis of the hydropathy index of 2 of these proteinsover the 20 amino acid sequence 281-300, inclusive of the lα7,β1 region,reveal that the amino acid substitutions in these proteins have made thelα7,β1 region much more hydrophobic. The grand average of hydropathyvalue (GRAVY) was determined for each protein sequence using thePC\GENE® (IntelliGenetics, Inc., Mountain View, Calif., release 6.85)protein sequence analysis computer program, SOAP, and a 7 amino acidinterval. The SOAP program is based on the method of Kyte and Doolittle(1982). The increase in hydrophobicity of the lα7,β1 region for eachprotein may increase the hydration of the loop and, therefore, theflexibility. The altered proteins, their respective amino acid changes,fold-increases over WT bioactivity, and GRAVY values are listed in Table9.

TABLE 9 HYDROPATHY VALUES FOR THE Lα7, β1 REGION OF CRY3BB AND 2DESIGNED CRY3BB PROTEINS SHOWING INCREASED SCRW BIOACTIVITY Amino FoldIncrease in Cry3Bb* Acid Bioactivity Over GRAVY Protein Changes WT(Amino Acids 281–300) wildtype — — 4.50 Cry3Bb.11241 Y287F, 2.6× 10.70D288N, R290L Cry3Bb.11242 R290V 2.5× 8.855.8.4 Alterations to Lβ1,α8 (Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230,Cry3Bb.11233, Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238 and Cry3Bb.11239)

The surface-exposed loop between β-strand 1 and α-helix 8 (lβ1,α8)defines the boundary between domains 1 and 2 of Cry3Bb. The introductionof semi-random amino acid changes to this region resulted in theidentification of several altered Cry3Bb proteins with increasedbioactivity. Hydropathy index analysis of the amino acid substitutionsfound in the altered proteins shows that the changes have made theexposed region more hydrophobic which may result in increased hydrationand flexibility. Table 10 lists the altered proteins, their respectiveamino acid changes and fold increases over WT Cry3Bb and the grandaverage of hydropathy value (GRAVY) determined using the PC\GENE®(IntelliGenetics, Inc., Mountain View, Calif., release 6.85) proteinsequence analysis prograrn, SOAP, over the 20 amino acid sequence305-324 inclusive of lβ1,α8 using a 7 amino acid interval.

TABLE 10 HYDROPATHY VALUES FOR THE Lβ1, α8 REGION OF CRY3BB AND 8DESIGNED CRY3BB* PROTEINS SHOWING INCREASED SCRW BIOACTIVITY FoldIncrease in GRAVY Cry3Bb* Amino Acid Bioactivity Over (Amino AcidsProtein Changes Wild Type 305–324) wildtype — — 0.85 Cry3Bb.11228 S311L,N313T, 4.1× 4.35 E317K Cry3Bb.11229 S311T, E317K, 2.5× 2.60 Y318CCry3Bb.11230 S311A, L312V, 4.7× 3.65 Q316W Cry3Bb.11233 S311A, Q316D2.2× 2.15 Cry3Bb.11236 S311I 3.1× 3.50 Cry3Bb.11237 S311I, N313H 5.4×3.65 Cry3Bb.11238 N313V, T314N, 2.6× 9.85 Q316M, E317V Cry3Bb.11239N313R, L315P, 2.8× 3.95 Q316L, E317A5.8.5 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242

Amino acid Q238, located in helix 6 of Cry3Bb, has been identified as aresidue that, by its large size and hydrogen bonding to R290, blockscomplete hydration of the space between helix 6 and helix 4.Substitution of R290 with amino acids that do not form hydrogen bonds orthat have side chains that can not span the physical distance tohydrogen bond with Q238 may result in increased hydration around Q238.Q238, unable to hydrogen bond to R290, may now bind water. This mayincrease the flexibility of the channel-forming region. Designedproteins Cry3Bb.11227 (R290N), Cry3Bb.11241 (R290L) and Cry3Bb.11242(R290V) show increased activities of approximately 2-fold, 2.6-fold and2.5-fold, respectively, against SCRW larvae compared to WT.

5.9 Example 9 Design Method 3: Manipulation of Hydrogen Bonds aroundMobile Regions

Mobility of regions of a protein may be required for activity. Themobility of the α5,6 region, the putative channel-forming region ofCry3Bb, may be improved by decreasing the number of hydrogen bonds,including salt bridges (hydrogen bonds between oppositely charged aminoacid side chains), between helices 5-6 and any other part of themolecule or dimer structure. These hydrogen bonds may impede themovement of the two helices. Decreasing the number of hydrogen bonds andsalt bridges may improve biological activity. Replacement ofhydrogen-bonding amino acids with hydrophobic residues must be done withcaution to avoid creating continuous hydrophobic surfaces betweenhelices 5-6 and any other part of the dimer. This may decrease mobilityby increasing hydrophobic surface interactions.

5.9.1 Cry3Bb.11222 and Cry3Bb.11223

Tyr230 is located on helix 6 and, in the quaternary dimer structure ofCry3Bb, this amino acid is coordinated with Tyr230 from the adjacentmolecule. Three hydrogen bonds are formed between the two helices 6 inthe two monomers because of this single amino acid. In order to improvethe flexibility of helices 5-6, the helices theoretically capable ofpenetrating the membrane and forming an ion channel, the hydrogen bondsacross the dimer were removed by changing this amino acid and acorresponding increase in biological activity was observed. The designedCry3Bb proteins, Cry3Bb.11222 and Cry3Bb.EG11223, show a 4-fold and2.8-fold increase in SCRW activity, respectively, compared to WT.

5.9.2 Cry3Bb.11051

Designed Cry3Bb protein Cry3Bb.11051 has amino acid change K189G inlα4,5 of domain 1. In the WT Cry3Bb structure, the exposed side chain ofK189 is close enough to the exposed side change of E123, located inlα2b,3, to form hydrogen bonds. Substitution of K189 with glycine, asfound in this position in Cry3A, removes the possibility of hydrogenbond formation at this site and results in a protein with a bioactivitythree-fold greater than WT Cry3Bb.

5.9.3 Cry3Bb.11227, Cry3Bb.11241 and Cry3Bb.11242

Amino acid Q238, located in helix 6 of Cry3Bb, has been identified as aresidue that, by its large size and hydrogen bonding to R290, blockscomplete hydration of the space between helix 6 and helix 4.Substitution of R290 with amino acids that do not form hydrogen bonds orthat have side chains that can not span the physical distance tohydrogen bond with Q238 may increase the flexibility of thechannel-forming region. Designed proteins Cry3Bb.11227 (R290N),Cry3Bb.11241 (R290L) and Cry3Bb.11242 (R290V) show increased activitiesof approximately 2-fold, 2.6-fold and 2.5-fold, respectively, againstSCRW larvae compared to WT

5.10 Example 10 Design Method 4: Loop Analysis and Loop Design aroundFlexible Helices

Loop regions of a protein structure may be involved in numerousfunctions of the protein including, but not limited to, channelformation, quaternary structure formation and maintenance, and receptorbinding. Cry3Bb is a channel-forming protein. The availability of theion channel-forming helices of δ-endotoxins to move into the bilayerdepend upon the absence of forces that hinder the process. One of theforces possibly limiting this process is the steric hindrance of aminoacid side chains in loop regions around the critical helices. Theliterature suggests that in at least one other bacterial toxin, not a B.thuringiensis toxin, the toxin molecule opens up or, in scientificterms, loses some of the quaternary structure to expose amembrane-active region (Cramer et al., 1990). This literature does notteach how to improve the probability of this event occurring and it isnot known if B. thuringiensis toxins use this same process to penetratethe membrane. Reducing the steric hindrance of the amino acid sidechains in these critical regions by reducing size or altering side chainpositioning with the corresponding increase in biological activity wasthe inventive step.

5.10.1 Analysis of the Loop between Helices 3 and 4 (Cry3Bb.11032)

The inventors have discovered that the first three helices of domain onecould be cleaved from the rest of the toxin by proteolytic digestion ofthe loop between helices α3 and α4 (Cry3Bb.60). Initial efforts totruncate the cry3Bb gene to produce this shortened, though more activeCry3Bb molecule, failed. For unknown reasons, B. thuringiensis failed tosynthesize this 60-kDa molecule. It was then reasoned that perhaps thefirst three helices of domain 1 did not have to be proteolyticallyremoved, or equivalently, the protein did not have to be synthesized inthis truncated form to take advantage of the Cry3Bb.60 design. It wasobserved that the protein Cry3A had a small amino acid near the lα3,4that might impart greater flexibility in the loop region therebypermitting the first three helices of domain 1 to move out of the way,exposing the membrane-active region. By designing a Cry3Bb molecule witha glycine residue near this loop, the steric hindrance of residues inthe loop might be lessened. The redesigned protein, Cry3Bb.11032, hasthe amino acid change D165G, which replaces the larger aspartate residue(average mass of 115.09) with the smallest amino acid, glycine (averagemass of 57.05). The activity of Cry3Bb.11032 is approximately 3-foldgreater than that of the WT protein. In this way, the loop betweenhelices α3 and α4 was rationally redesigned with a correspondingincrease in the biological activity.

5.10.2 Cry3Bb.11051

The loop region connecting helices α4 and α5 in Cry3Bb must be flexibleso that the channel-forming helices α5-α6 can penetrate into themembrane. It was noticed that Cry3A has a glycine residue in the middleof this loop that may impart greater flexibility. The correspondingchange, K189G, was made in Cry3Bb and the resulting, designed protein,Cry3Bb.11051, exhibits a 3-fold increase in activity against SCRW larvaecompare to WT Cry3Bb.

5.10.3 Analysis of the Loop between β-Strand 1 and Helix 8(Cry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239)

The loop region located between β strand 1 of domain 2 and α helix 8 indomain 2 is very close to the loop between α helices 6 and 7 indomain 1. Some of the amino acids side chains of lβ1,α8 appear as thoughthey may sterically impede movement of lα6,7. Since lα6,7 must beflexible for the channel-forming helices α5-α6 to insert into themembrane, it was thought that re-engineering this loop may change thepositioning of the side chains resulting in less steric hindrance. Thiswas accomplished creating proteins with increased biological activitiesranging from 2.2 to 5.4 times greater than WT. These designed toxinproteins and their amino acid changes are listed in Table 2 asCry3Bb.11228, Cry3Bb.11229, Cry3Bb.11230, Cry3Bb.11232, Cry3Bb.11233,Cry3Bb.11236, Cry3Bb.11237, Cry3Bb.11238, and Cry3Bb.11239.

5.10.4 Analysis of the Loop between Helix 7 and β-Strand 1(Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241, Cry3Bb.11242, andCry3Bb.11036)

If Cry3Bb is similar to a bacterial toxin which must open up to expose amembrane active region for toxicity, it is possible that other helicesin addition to the channel-forming helices must also change positions.It was reasoned that, if helices α5-α6 insert into the membrane, thanhelix α7 may have to change positions also. It was shown in example4.4.3 that increasing flexibility between helix α6 and α7 can increaseactivity, greater flexibility in the loop following helix α7, lα7,β1 mayalso increase bioactivity. Alterations to the lα7,β1 region of Cry3Bbresulted in the isolation of several proteins with increased activitiesranging from 1.9 to 4.3 times greater than WT. These designed proteinsare listed in Table 7 as Cry3Bb.11227, Cry3Bb.11234, Cry3Bb.11241,Cry3Bb.11242, and Cry3Bb.11036.

5.11 Example 11 Design Method 5: Loop Design around β Strands and βSheets

Loop regions of a protein structure may be involved in numerousfunctions of the protein including, but not limited to, channelformation, quaternary structure formation and maintenance, and receptorbinding. A binding surface is often defined by a number of loops, as isthe case with immunoglobulin G (IgG) (see Branden and Tooze, 1991, forreview). What can not be determined at this point, however, is whatloops will be important for receptor interactions just by looking at thestructure of the protein in question. Since a receptor has not beenidentified for Cry3Bb, it is not even possible to compare the structureof Cry3Bb with other proteins that have the same receptor for structuralsimilarities. To identify Cry3Bb loops that contribute to receptorinteractions, random mutagenesis was performed on surface-exposed loops.

As each loop was altered, the profile of the overall bioactivities ofthe resultant proteins were examined and compared. The loops, especiallyin domain 2 which appears to be unnecessary for channel activity, fallinto two categories: (1) loops that could be altered without much changein the level of bioactivity of the resultant proteins and (2) loopswhere alterations resulted in overall loss of resultant proteinbioactivity. Using this design method, it is possible to identifyseveral loops important for activity.

5.11.1 Analysis of Loop β 2,3

Semi-random mutagenesis of the loop region between β strands 2 and 3resulted in the production of structurally stable toxin proteins withsignificantly reduced activities against SCRW larvae. The lβ2,3 regionis highly sensitive to amino acid changes indicating that specific aminoacids or amino acid sequences are necessary for toxin protein activity.It is conceivable, therefore, that specific changes in the lβ2,3 regionwill increase the binding and, therefore, the activity of the redesignedtoxin protein.

5.11.2 Analysis of Loop β 6,7

Semi-random mutations introduced to the loop region between β strands 6and 7 resulted in structurally stable proteins with an overall loss ofSCRW bioactivity. The lβ6,7 region is highly sensitive to amino acidchanges indicating that specific amino acids or amino acid sequences arenecessary for toxin protein activity. It is conceivable, therefore, thatspecific changes in the lβ6,7 region will increase the binding and,therefore, the activity of the redesigned toxin protein.

5.11.3 Analysis of Loop β 10,11

Random mutations to the loop region between β strands 10 and 11 resultedin proteins having an overall loss of SCRW bioactivity. Loop β10,11 isstructurally close to and interacts with loops β2,3 and β6,7. Specificchanges to individual residues within the lβ10,11 region may also resultin increased interaction with the insect membrane, increasing thebioactivity of the toxin protein.

5.11.4 Cry3Bb.11095

Loops β2,3, β6,7 and β10,11 have been identified as important forbioactivity of Cry3Bb. The 3 loops are surface-exposed and structurallyclose together. Amino acid Q348 in the WT structure, located in β-strand2 just prior to lβ2,3, does not form any intramolecular contacts.However, replacing Q348 with arginine (Q348R) results in the formationof 2 new hydrogen-bonds between R348 and the backbone carbonyls of R487and R488, both located in lβ10,11. The new hydrogen bonds may act tostabilize the structure formed by the 3 loops. The designed proteincarrying this change, Cry3Bb.11095, is 4.6-fold more active than WTCry3Bb.

5.12 Example 12 Design Method 6: Identification and Re-Design of ComplexElectrostatic Surfaces

Interactions of proteins include hydrophobic interactions (e.g., Van derWaals forces), hydrophilic interactions, including those betweenopposing charges on amino acid side chains (salt bridges), and hydrogenbonding. Very little is known about δ-endotoxin and receptorinteractions. Currently, there are no literature reports identifying thetypes of interactions that predominate between B. thuringiensis toxinsand receptors.

Experimentally, however, it is important to increase the strength of theB. thuringiensis toxin-receptor interaction and not permit the precisedetermination of the chemical interaction to stand in the way ofimproving it. To accomplish this, the electrostatic surface of Cry3Bbwas defined by solving the Poisson-Boltzman distribution around themolecule. Once this electrically defined surface was solved, it couldthen be inspected for regions of greatest diversity. It was reasonedthat these electrostatically diverse regions would have the greatestprobability of participating in the specific interactions between the B.thuringiensis toxin proteins and the receptor, rather than more generaland non-specific interactions. Therefore, these regions were chosen forredesign, continuing to increase the electrostatic diversity of theregions. In addition, examination of the electrostatic interactionaround the putative channel forming region of the toxin created insightsfor redesign. This includes identification of an electropositive residuein an otherwise negatively charged conduit (see example 4.6.1).

5.12.1 R290 (Cry3Bb.11227, Cry3Bb.11241, and Cry3Bb.11242)

Examination of the Cry3Bb dimer interface along the domain 1 axissuggested that a pore or conduit for cations might be formed between themonomers. Electrostatic examination of this axis lent additionalcredibility to this suggestion. In fact, the hypothetical conduit isprimarily negatively charged, an observation consistent with thebiophysical analysis of cation-selective, δ-endotoxin channels. If acation channel were formed along the axis of the dimer, then the cationcould move between the monomers relatively easily with only onesignificant hurdle. A positively charged arginine residue (R290) lies inthe otherwise negatively charged conduit. This residue could impede thecation movement through the channel. Based on this analysis, R290 waschanged to uncharged residues. The bioactivity of redesigned proteinsCry3Bb.11227 (R290N), Cry3Bb.11241 (R290L) and Cry3Bb.11242 (R290V) wasimproved approximately 2-fold, 2.6-fold and 2.5-fold, respectively.

5.12.2 Cry3Bb.60

Trypsin digestion of solubilized Cry3Bb yields a stable, truncatedprotein with a molecular weight of 60 kDa (Cry3Bb.60). Trypsin digestionoccurs on the carboxyl side of residue R159, effectively removinghelices 1 through 3 from the native Cry3Bb structure. The cleavage ofthe first 3 helices exposes an electrostatic surface different thanthose found in the native structure. The new surface has a combinationof hydrophobic, polar and charged characteristics that may play a rolein membrane interactions. The bioactivity of Cry3Bb.60 is 3.6-foldgreater than that of WT Cry3Bb.

5.13 Example 13 Design Method 7: Identification and Removal of MetalBinding Sites

The literature teaches that the in vitro behavior of B. thuringiensistoxins can be increased by chelating divalent cations from theexperimental system (Crawford and Harvey 1988). It was not known,however, how these divalent cations inhibited the in vitro activity.Crawford and Harvey (1988) demonstrated that the short circuit currentacross the midgut was more severely inhibited by B. thuringiensis in thepresence of EDTA, a chelator of divalent ions, than in the absence ofthis agent, thus suggesting that this step in the mode of action of B.thuringiensis could be potentiated by removing divalent ions. Similarobservations were made using black-lipid membranes and measuring anincrease in the current created by the δ-endotoxins in the presence ofEDTA to chelate divalent ions. There were at least three possibleexplanations for these observations. The first explanation could be thatthe divalent ions are too large to move through a ion channel moresuitable for monovalent ions, thereby blocking the channel. Second, thedivalent ions may cover the protein in the very general way, therebybuffering the charge interactions required for toxin membraneinteraction and limiting ion channel activity. The third possibility isthat a specific metal binding site exists on the protein and, whenoccupied by divalent ions, the performance of the ion channel isimpaired. Although the literature could not differentiate the value ofone possibility over another, the third possibility led to an analysisof the Cry3Bb structure searching for a specific metal binding site thatmight alter the probability that a toxin could form an ion channel.

5.13.1 H231 (Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225, and Cry3Bb.11226)

A putative metal binding site is formed in the Cry3Bb dimer structure bythe H231 residues of each monomer. The H231 residues, located in helixα6, lie adjacent to each other and close to the axis of symmetry of thedimer. Removal of this site by replacement of histidine with other aminoacids was evaluated by the absence of EDTA-dependent ion channelactivity. The bioactivities of the designed toxin proteins,Cry3Bb.11222, Cry3Bb.11224, Cry3Bb.11225 and Cry3Bb.11226, are increased4-, 5-, 3.6- and 3-fold, respectively, over that of WT Cry3Bb. Theirrespective amino acid changes are listed in Table 2.

5.14 Example 14 Design Method 8: Alteration of Quaternary Structure

Cry3Bb can exist in solution as a dimer similar to a related protein,Cry3A (Walters et al., 1992). However, the importance of the dimer tobiological activity is not known because the toxin as a monomer or as ahigher order structure has not been seriously evaluated. It is assumedthat specific amino acid residues contribute to the formation andstability of the quaternary structure. Once a contributing residue isidentified, alterations can be made to diminish or enhance the effect ofthat residue thereby affecting the interaction between monomers. Channelactivity is a useful way, but by no means the only way, to assessquaternary structure of Cry3Bb and its derivatives. It has been observedthat Cry3Bb creates gated conductances in membranes that grow in sizewith time, ultimately resulting in large pores in the membrane (thechannel activity of WT Cry3Bb is described in Section 12.1). It also hasbeen observed that Cry3A forms a more stable dimer than Cry3Bb andcoincidentally forms higher level conductances faster (FIG. 10). Thisobservation led the inventors to propose that oligomerization and ionchannel formation (conductance size and speed of channel formation) wererelated. Based on this observation Cry3Bb was re-engineered to makelarger and more stable oligomers at a faster rate. It is assumed in thisanalysis that the rate of ion channel formation and growth mirrors thisprocess. It is also possible that changes in quaternary structure maynot affect channel activity alone or at all. Alterations to quaternarystructure may also affect receptor interactions, protein processing inthe insect gut environment, as well as other aspects of bioactivityunknown.

5.14.1 Cry3Bb.11048

Comparative structural analysis of Cry3A and Cry3Bb led to theidentification of structural differences between the two toxins in theion channel-forming domain; specifically, an insertion of one amino acidbetween helix 2a and helix 2b in Cry3Bb. Removal of this additionalamino acid in Cry 3B2, A104, and a D103E substitution, as in Cry3A,resulted in loss of channel gating and the formation of symmetricalpores. Once the pores are formed they remain open and allow a steadyconductance ranging from 25-130 pS. This designed protein, Cry3Bb.11048,is 4.3 times more active than WT Cry3Bb against SCRW larvae.

5.14.2 Oligomerization of Cry3Bb.60

Individual molecules of Cry3Bb or Cry3Bb.60 can form a complex withanother like molecule. Oligomerization of Cry3Bb is demonstrated bySDS-PAGE, where samples are not heated in sample buffer prior to loadingon the gel. The lack of heat treatment allows some nondenatured toxin toremain. Oligomerization is visualized following Coomassie staining bythe appearance of a band at 2 times the molecular weight of the monomer.The intensity of the higher molecular weight band reflects the degree ofoligomerization. The ability of Cry3Bb to form an oligomer is notreproducibly apparent. The complex cannot be repeatedly observed toform. Cry3Bb.60, however, forms a significantly greater amount of ahigher molecular weight complex (120 kDa). These data suggest thatCry3Bb.60 more readily forms the higher order complex than Cry3Bb alone.Cry3Bb.60 also forms ion channels with greater frequency than WT Cry3Bb(see Section 5.12.9).

5.14.3 Cry3Bb.11035

Changes were made in Cry3Bb to reflect the amino acid sequence in Cry3Aat the end of lα3,4 and in the beginning of helix 4. These changesresulted in the designed protein, Cry3Bb.11035, that, unlike wild typeCry3Bb, forms spontaneous channels with large conductances. Cry3Bb.11035is also approximately three times more active against SCRW larvae thanWT Cry3Bb. Cry3Bb.11035 and its amino acid changes are listed in Table10.

5.14.4 Cry3Bb.11032

Cry3Bb.11032 was altered at residue 165 in helix α4, changing anasparate to glycine, as found in Cry3A. Cry3Bb.11032 is three-fold moreactive than WT Cry3Bb. The channel activity of Cry3Bb.11032 is much likeCry3Bb except when the designed protein is artificially incorporatedinto the membrane. A 16-fold increase in the initial channelconductances is observed compared to WT Cry3Bb (see Section 5.12.2).This increase in initial conductance presumably is due to enhancedquaternary structure, stability or higher-order structure.

5.14.5 EG11224

In the WT Cry3Bb dimer structure, histidine, at position 231 in domain1, makes hydrogen bond contacts with D288 (domain 1), Y230 (domain 1),and, through a network of water molecules, also makes contacts to D610(domain 3), all of the opposite monomer. D610 and K235 (domain 1) alsomake contact. Replacing the histidine with an arginine, H231R, results,in one orientation, in the formation of a salt bridge to D610 of theneighboring monomer. In a second orientation, the contacts with D288 ofthe neighboring monomer, as appear in the WT structure, are retained. Ineither orientation, R231 does not hydrogen bond to Y230 of the oppositemonomer but does make contact with K235 which retains is contacts toK610 (V. Cody, research communication). The shifting hydrogen bonds havechanged the interactions between the different domains of the protein inthe quaternary structure. Overall, fewer hydrogen bonds exist betweendomains 1 of the neighboring monomers and a much stronger bond has beenformed between domains 1 and 3. Channel activity was found to bealtered. Cry3Bb.11224 produces small, quickly gating channels likeCry3Bb. However, unlike WT Cry3Bb, Cry3Bb.11224 does not exhibitβ-mercaptoethanol-dependent activation. Replacing H231 with arginineresulted in a designed Cry3Bb protein, Cry3Bb.11224, exhibiting a 5-foldincrease in bioactivity.

5.14.6 Cry3Bb.11226

Cry3Bb.11226 is similar to Cry3Bb.11224, discussed in Section 4.8.5, inthat the histidine at position 231 has been replaced. The amino acidchange, H231T, results in the loss of β-mercaptoethanol dependentactivation seen with WT Cry3Bb (see Section 5.12.1). The replacement ofH231, a putative metal binding site, changes the interaction of regionsin the quaternary structure resulting in a different type of channelactivity. Cry3Bb.11226 is three-fold more active than WT Cry3Bb.

5.14.7 Cry3Bb.11221

Cry3Bb.11221 has been re-designed in the lα3,4 region of Cry3Bb. Thechannels formed by Cry3Bb.11221 are much more well resolved than theconductances formed by WT Cry3Bb (see Section 5.12.6). Cry3Bb.11221exhibits a 6.4-fold increase in bioactivity over that of WT Cry3Bb. Theamino acid changes found in Cry3Bb.11221 are listed in Table 2.

5.14.8 Cry3Bb.11242

The designed protein, Cry3Bb.11242, carrying the alteration R290V, formssmall conductances immediately which grow rapidly and steadily to largeconductances in about 3 min (see Section 5.12.7). This is contrast to WTCry3Bb channels which take 30-45 min to appear and grow slowly overhours to large conductances. Cry3Bb.11242 also exhibits a 2.5-foldincrease in bioactivity compared to WT Cry3Bb.

5.14.9 Cry3Bb.11230

Cry3Bb.11230, unlike WT Cry3Bb, forms well resolved channels with longopen states. These channels reach a maximum conductance of 3000 pS butdo not continue to grow with time. Cry3Bb.11230 has been re-designed inthe lβ1,α8 region of Cry3Bb and exhibits almost a 5-fold increase inactivity against SCRW larvae (Table 9) and a 5.4-fold increase againstWCRW larvae (Table 10) compared to WT Cry3Bb. The amino acid changesfound in Cry3Bb.11230 are listed in Table 2.

5.15 Example 15 Design Method 9: Design of Structural Residues

The specific three-dimensional structure of a protein is held in placeby amino acids that may be buried or otherwise removed from the surfaceof the protein. These structural determinants can be identified byinspection of forces responsible for the surface structure positioning.The impact of these structural residues can then be enhanced to restrictmolecular motion or diminished to enhance molecular flexibility.

5.15.1 Cry3Bb.11095

Loops β2,3, β6,7 and β10,11, located in domain 2 of Cry3Bb, have beenidentified as important for bioactivity. The three loops aresurface-exposed and structurally close together. Amino acid Q348 in theWT structure, located in β-strand 2 just prior to lβ2,3, does not formany intramolecular contacts. However, replacing Q348 with arginine(Q348R) results in the formation of 2 new hydrogen-bonds between R348and the backbone carbonyls of R487 and R488, both located in lβ10,11.The new hydrogen bonds may act to stabilize the structure formed by thethree loops. Certainly, the structure around R348 is more tightly packedas determined by X-ray crystallography. The designed protein carryingthis change, Cry3Bb.11095, is 4.6-fold more active than WT Cry3Bb.

5.16 Example 16 Design Method 10: Combinatorial Analysis and Mtuagenesis

Individual sites in the engineered Cry3Bb molecule can be used togetherto create a Cry3Bb molecule with activity even greater than the activityof any one site. This method has not been precisely applied to anyδ-endotoxin. It is also not obvious that improvements in two sites canbe pulled together to improve the biological activity of the protein. Infact, data demonstrates that improvements to 2 sites, when pulledtogether into a single construct, do not necessarily further improve thebiological activity of Cry3Bb. In some cases, the combination resultedin decreased protein stability and/or activity. Examples of proteinswith site combinations that resulted in improved activity compared to WTCry3Bb but decreased activity compared to 1 or more of the “parental”proteins are Cry3Bb.11235, 11046, 11057 and 11058. Cry3Bb.11082, whichcontains designed regions from 4 parental proteins, retains the level ofactivity from the most active parental strain (Cry3Bb.11230) but doesnot show an increase in activity. These proteins are listed in Table 7.The following are examples of instances where combined mutations havesignificantly improved biological activity.

5.16.1 Cry3Bb.11231

Designed protein Cry3Bb.11231 contains the alterations found inCry3Bb.11224 (H231R) and Cry3Bb.11228 (changes in lβ1,α8). Thecombination of amino acid changes found in Cry3Bb.11231 results in anincrease in bioactivity against SCRW larvae of approximately 8-fold overthat of WT Cry3Bb (Table 2). This increase is greater than exhibited byeither Cry3Bb.11224 (5.0×) or Cry3Bb.11228 (4.1×) alone. Cry3Bb.11231was also exhibits an 12.9-fold increase in activity compared to WTCry3Bb against WCRW larvae (Table 10).

5.16.2 Cry3Bb.11081

Designed Cry3Bb protein Cry3Bb.11081 was constructed by combining thechanges found in Cry3Bb.11032 and Cry3Bb.11229 (with the exception ofY318C). Cry3Bb.11081 a 6.1-fold increase in activity over WT Cry3Bb; agreater increase in activity than either of the individual parentalproteins, Cry3Bb.11032 (3.1-fold) and Cry3Bb.11229 (2.5-fold).

5.16.3 Cry3Bb.11083

Designed Cry3Bb protein Cry3Bb.11083 was constructed by combining thechanges found in Cry3Bb.11036 and Cry3Bb.11095. Cry3Bb.11083 exhibits a7.4-fold increase in activity against SCRW larvae compared to WT Cry3Bb;a greater increase than either Cry3Bb.11036 (4.3×) or Cry3Bb.11095(4.6×). Cry3Bb.11083 also exhibits a 5.4-fold increase in activityagainst WCRW larvae compared to WT Cry3Bb (Table 10).

5.16.4 Cry3Bb.11084

Designed Cry3Bb protein Cry3Bb.11084 was constructed by combining thechanges found in Cry3Bb.11032 and the S311L change found inCry3Bb.11228. Cry3Bb.11084 exhibits a 7.2-fold increase in activity overthat of WT Cry3Bb; a greater than either Cry3Bb.11032 (3.1×) orCry3Bb.11228 (4.1×).

5.16.5 Cry3Bb.11098

Designed Cry3Bb protein Cry3Bb.11098 was constructed to contain thefollowing amino acid changes: D165G, H231R, S311L, N313T, and E317K. Thenucleic acid sequence is given in SEQ ID NO:107, and the encoded aminoacid sequence is given in SEQ ID NO:108.

5.17 Example 17 Design Strategy 11: Alteration of Binding toGlycoproteins and to WCRW Brush Border Membranes

While the identity of receptor(s) for Cry3Bb is unknown, it isnonetheless important to increase the interaction of the toxin with itsreceptor. One way to improve the toxin-receptor interaction with knowingthe identity of the receptor is to reduce or eliminate non-productivebinding to other biomolecules. The inventors have observed that Cry3Bbbinds non-specifically to bovine serum albumin (BSA) that has beenglycosylated with a variety of sugar groups, but not to non-glycosylatedBSA. Cry3A, which is not active on Diabrotica species, shows similar buteven greater binding to glycosylated-BSA. Similarly, Cry3A shows greaterbinding to immobolized WCRW brush border membrane (BBM) than does WTCry3Bb, suggesting that much of the observed binding is non-productive.It was reasoned that the non-specific binding to WCRW BBM occurs viaglycosylated proteins, and that binding to both glycosylated-BSA andWCRW BBM is non-productive in reaction pathway to toxicity. Thereforereduction or elimination of that binding would lead to enhanced bindingto the productive receptor and to enhanced toxicity. Potential bindingsites for sugar groups were targeted for redesign to reduce thenon-specific binding of Cry3Bb to glycoproteins and to immobilized WCRWBBM.

5.17.1 Cry3Bb.60

Cry3Bb-60, in which Cry3Bb has been cleaved at R159 in lα3,4, showsdecreased binding to glycosylated-BSA and decreased binding toimmobilized WCRW BBM. Cry3Bb-60 shows a 3.6-fold increase in bioactivityrelative to WT Cry3Bb.

5.17.2 Alterations to lα3,4 (Cry3Bb.11221)

Cry3Bb.11221 has been redesigned in the lα3,4 region of domain 1, whichis the region in which Cry3Bb is cleaved to produce Cry3Bb-60.Cry3Bb.11221 also shows decreased binding to both glycosylated-BSA andimmobilized WCRW BBM, and exhibits a 6.4-fold increase in bioactivityover that of WT Cry3Bb. Together with data for Cry3Bb.60 (section5.17.1) these data suggest that this loop region contributessubstantially to non-productive binding of the toxin.

5.17.3 Alteration of lβ1,α8 (Cry3Bb.11228,11230,11237 and 11231)

The lβ1,α8 region of Cry3Bb has been re-engineered to increase hydration(section 4.2.4) and enhance flexibility (section 4.4.3). Severalproteins altered in this region, Cry3Bb.11228, 11230, and 11237demonstrate substantially lower levels of binding both glycosylated-BSAand immobilized WCRW BBM, and also show between 4.1- and 4.5-foldincreases in bioactivity relative to WT Cry3Bb.

5.17.4 Binding Activity

The tendencies of Cry3Bb and some of its derivatives to bind toglycosylated-BSA and to WCRW BBM were determined using a BIAcore™surface plasmon resonance biosensor. For glycosylated-BSA binding, theglycosylated protein was immobilized using standard NHS chemistry to aCM5 chip (BIAcore), and the solubilized toxin was injected over theglycosylated-BSA surface. To measure binding to WCRW BBM, brush bordermembrane vesicles (BBMV) purified from WCRW midguts (English et al.,1991) were immobilized on an HPA chip (BIAcore) then washed with either10 mM KOH or with 40 mM β-octylglucoside. The solubilized toxin was theninjected over the resulting hybrid bilayer surface to detect binding.Protein concentration were determined by Protein Dye Reagent assay(BioRad) or BCA Protein Assay (Pierce). Other methods may also be usedto determine the same binding information. These include, but are notlimited to, ligand blot experiments using labeled toxin, labeledglycosylated protein, or anti-toxin antibodies, affinity chromatography,and in vitro binding of toxin to intact BBMV.

5.18 Example 18 Construction of Plasmids with WT Cry3Bb Sequences

Standard recombinant DNA procedures were performed essentially asdescribed by Sambrook et al., (1989).

5.18.1 pEG1701

pEG1701 (FIG. 11), contained in EG11204 and EG11037, was constructed byinserting the SphI-PstI fragment containing the cry3Bb gene and thecry1F terminator from pEG911 (Baum, 1994) into the SphI-PstI site ofpEG854.9 (Baum et al., 1996), a high copy number B. thuringiensis-E.coli shuttle vector.

5.18.2 pEG1028

pEG1028 contains the HindIII fragment of cry3Bb from pEG1701 cloned intothe multiple cloning site of pTZ18U at HindIII.

5.19 Example 19 Construction of Plasmids with Altered Cry3Bb Genes

Plasmid DNA from E. coli was prepared by the alkaline lysis method(Maniatis et al., 1982) or by commercial plasmid preparation kits(examples: PERFECTprep™ kit, 5 Prime-3 Prime, Inc., Boulder Colo.;QIAGEN plasmid prep kit, QIAGEN Inc.). B thuringiensis plasmids wereprepared from cultures grown in brain heart infusion plus 0.5% glycerol(BHIG) to mid logarithmic phase by the alkaline lysis method. Whennecessary for purification, DNA fragments were excised from an agarosegel following electrophoresis and recovered by glass milk using aGeneclean II® kit (BIO 101 Inc., La Jolla, Calif.). Alteration of thecry3Bb gene was accomplished using several techniques includingsite-directed mutagenesis, triplex PCR™, quasi-random PCR™ mutagenesis,DNA shuffling and standard recombinant techniques. These techniques aredescribed in Sections 6.1, 6.2, 6.3, 6.4 and 6.5, respectively. The DNAsequences of primers used are listed in Section 7.

5.20 Example 20 Site-Directed Mutagenesis

Site-directed mutagenesis was conducted by the protocols established byKunkle (1985) and Kunkle et al. (1987) using the Muta-Gene™ M13 in vitromutagenesis kit (Bio-Rad, Richmond, Calif.). Combinations of alterationsto cry3Bb were accomplished by using the Muta-Gene™ kit and multiplemutagenic oligonucleotide primers.

5.20.1 pEG1041

pEG1041, contained in EG11032, was constructed using the Muta-Gene™ kit,primer C, and single-stranded pEG1028 as the DNA template. The resultingaltered cry3Bb DNA sequence was excised as a PflMI DNA fragment and usedto replace the corresponding DNA fragment in pEG1701.

5.20.2 pEG1046

pEG1046, contained in EG11035, was constructed using the Muta-Gene™ kit,primer D, and single-stranded pEG1028 as the DNA template. The resultingaltered cry3Bb DNA sequence was excised as a PflMI DNA fragment and usedto replace the corresponding DNA fragment in pEG1701.

5.20.3 pEG1047

pEG1047, contained in EG11036, was constructed using the Muta-Gene™ kit,primer E, and single-stranded pEG1028 as the DNA template. The resultingaltered cry3Bb DNA sequence was excised as a PflMI DNA fragment and usedto replace the corresponding DNA fragment in pEG1701.

5.20.4 pEG1052

pEG1052, contained in EG11046, was constructed using the Muta-Gene™ kit,primers D and E, and single-stranded pEG1028 as the DNA template. Theresulting altered cry3Bb DNA sequence was excised as a PflMI DNAfragment and used to replace the corresponding DNA fragment in pEG1701.

5.20.5 pEG1054

pEG1054, contained in EG11048, was constructed using the Muta-Gene™ kit,primer F, and single-stranded pEG1028 as the DNA template. The resultingaltered cry3Bb DNA sequence was excised as a PflMI DNA fragment and usedto replace the corresponding DNA fragment in pEG1701.

5.20.6 pEG1057

pEG1057, contained in EG11051, was constructed using the Muta-Gene™ kit,primer G, and single-stranded pEG1028 as the DNA template. The resultingaltered cry3Bb DNA sequence was excised as a PflMI DNA fragment and usedto replace the corresponding DNA fragment in pEG1701.

5.21 Example 21 Triplex PCR™

Triplex PCR™ is described by Michael (1994). This method makes use of athermostable ligase to incorporate a phosphorylated mutagenic primerinto an amplified DNA fragment during PCR™. PCR™ was performed on aPerkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer, Norwalk, Conn.)using a AmpliTaq™ DNA polymerase kit (Perkin-Elmer) and SphI-linearizedpEG1701 as the template DNA. PCR™ products were cleaned using commercialkits such as Wizard™ PCR™ Preps (Promega, Madison, Wis.) and QIAquickPCR™ Purification kit (QIAGEN Inc., Chatsworth, Calif.).

5.21.1 pEG1708 and pEG1709

pEG1708 and pEG1709, contained in EG11222 and EG11223, respectively,were constructed by replacing the PflMI-PflMI fragment of cry3Bb inpEG1701 with PflMI-digested and gel purified PCR™ fragment altered atcry3Bb nucleotide positions 688-690. encoding amino acid Y230. Randommutations were introduced into the Y230 codon by triplex PCR™. Mutagenicprimer MVT095 was phosphorylated and used together with outside primerpair FW001 and FW006. Primer MVT095 also contains a silent mutation atposition 687, changing T to C, which, upon incorporation, introduces anadditional EcoRI site into pEG1701.

5.21.2 pEG1710 pEG1711 and pEG1712

Plasmids pEG1710, pEG1711 and pEG1712, contained in EG11224, EG11225 andEG11226, respectively, were created by replacing the PflMI-PflMIfragment of the cry3Bb gene in pEG1701 with PflMI-digested and gelpurified PCR™ fragment altered at cry3Bb nucleotide positions 690-692,encoding H231. Random mutations were introduced into the H231 codon bytriplex PCR™. Mutagenic primer MVT097 was phosphorylated and usedtogether with outside primer pair FW001 and FW006. Primer MVT097 alsocontains a T to C sequence change at position 687 which, uponincorporation, results in an additional EcoRI site by silent mutation.

5.21.3 pEG1713 and pEG1727

pEG1713 and pEG1727, contained in EG11227 and EG11242, respectively,were constructed by replacing the PflMI-PflMI fragment of the cry3Bbgene in pEG1701 with PflMI-digested and gel purified PCR™ fragmentaltered at cry3Bb nucleotide positions 868-870, encoding amino acidR290. Triplex PCR™ was used to introduce random changes into the R290codon. The mutagenic primer, MVT091, was designed so that the nucleotidesubstitutions would result in approximately 36% of the sequencesencoding amino acids D or E. MVT091 was phosphorylated and used togetherwith outside primer pair FW001 and FW006.

5.22 Example 22

Quasi-Random PCR™ Mutagenesis

Quasi-random mutagenesis combines the mutagenic PCR™ techniquesdescribed by Vallette et al. (1989), Tomic et al. (1990) and LaBean andKauffman (1993). Mutagenic primers, sometimes over 70 nucleotides inlength, were designed to introduce changes over nucleotide positionsencoding for an entire structural region, such as a loop. Degeneratecodons typically consisted of a ratio of 82% WT nucleotide plus 6% eachof the other 3 nucleotides per position to semi-randomly introducechanges over the target region (LaBean and Kauffman, 1993). Whenpossible, natural restriction sites were utilized; class 2s enzymes wereused when natural sites were not convenient (Stemmer and Morris, 1992,list additional restriction enzymes useful to this technique). PCR™ wasperformed on a Perkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer,Norwalk, Conn.) using a AmpliTaq™ DNA polymerase kit (Perkin-Elmer) andSphI-linearized pEG1701 as the template DNA. Quasi-random PCR™amplification was performed using the following conditions: denaturationat 94° C. for 1.5 min.; annealing at 50° C. for 2 min. and extension at72° C. for 3 min., for 30 cycles. The final 14 extension cycles wereextended an additional 25 s per cycle. Primers concentration was 20 μMper reaction or 40 μM for long, mutagenic primers. PCR™ products werecleaned using commercial kits such as Wizard™ PCR™ Preps (Promega,Madison. Wis.) and QIAquick PCR™ Purification kit (QIAGEN Inc.,Chatsworth, Calif.). In some instances PCR™ products were treated withKlenow Fragment (Promega) following the manufacturer's instructions tofill in any single base overhangs prior to restriction digestion.

5.22.1 pEG1707

EG1707, contained in EG11221, was constructed by replacing thePflMI-PflMI fragment of the cry3Bb gene in pEG1701 with PflMI-digestedand gel purified PCR™ fragment altered at cry3Bb nucleotide positions460-480, encoding lα3,4 amino acids 154-160. Primer MVT075, whichincludes a recognition site for the class 2s restriction enzyme BsaI,and primer FW006 were used to introduce changes into this region byquasi-random mutagenesis. Primers MVT076, also containing a BsaI site,and primer FW001 were used to PCR™ amplify a “linker” fragment.Following PCR™ amplification, both products were cleaned, end-filled,digested with BsaI and ligated to each other. Ligated fragment was gelpurified and used as template for PCR™ amplification using primer pairFW001 and FW006. PCR™ product was cleaned, digested with PflMI, gelpurified and ligated into PflMI-digested and purified pEG1701 vectorDNA.

5.22.2 pEG1720 and pEG1726

pEG1720 and pEG1726, contained in EG11234 and EG11241, respectively,were constructed by replacing the PflMI-PflMI fragment of the cry3Bbgene in pEG1701 with PflMI-digested and gel purified PCR™ fragmentaltered at cry3Bb nucleotide positions 859-885, encoding lα7,β1 aminoacids 287-295. Quasi-random PCR™ mutagenesis was used to introducechanges into this region. Mutagenic primer MVT111, designed with a BsaIsite, and primer FW006 were used to introduce the changes. Primer pairMVT094, also containing a BsaI site, and FW001 were used to amplify thelinker fragment. The PCR™ products were digested with BsaI, gel purifiedthen ligated to each other. Ligated product was PCR™ amplified usingprimer pair FW001 and FW006, digested with PflMI.

5.22.3 pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722, pEG1723,pEG1724 and pEG1725

pEG1714, pEG1715, pEG1716, pEG1718, pEG1719, pEG1722, pEG1723, pEG1724and pEG1725, contained in EG11228, EG11229, EG11230, EG11232, EG11233,EG11236, EG11237, EG11238 and EG11239, respectively, were constructed byreplacing the PflMI-PflMI fragment of the cry3Bb gene in pEG1701 withPflMI-digested and gel purified PCR™ fragment altered at cry3Bbnucleotide positions 931-954, encoding lβ1,α8 amino acids 311-318.Quasi-random PCR™ mutagenesis was used to introduce changes into thisregion using mutagenic primer MVT103 and primer FW006. Primers FW001 andFW006 were used to amplify a linker fragment. The PCR™ products wereend-filled using Klenow and digested with BamHI. The larger fragmentfrom the FW001-FW006 digest was gel purified then ligated to thedigested MVT103-FW006 fragment. Ligated product was gel purified andamplified by PCR™ using primer pair FW001 and FW006. The amplifiedproduct was digested with PflMI and gel purified prior to ligation intoPflMI-digested and purified pEG1701 vector DNA.

5.22.4 pEG1701.lβ2.3

Plasmids carrying alterations of cry3Bb WT sequence at nucleotides1051-1065, encoding structural region lβ2,3 of Cry3Bb, were constructedby replacing the MluI-SpeI fragment of pEG1701 with isolated MluI- andSpeI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis were mutagenic primer MVT081 was pairedwith FW006. These plasmids as a group are designated pEG1701.1β2,3.

5.22.5 pEG1701.lβ6,7

Plasmids containing mutations of the cry3Bb WT sequence at nucleotides1234-1248, encoding structural region lβ6,7 of Cry3Bb, were constructedby replacing the MluI-SpeI fragment of pEG1701 with isolated MluI- andSpeI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis where mutagenic primer MVT085 was pairedwith primer WD115. Primer pair MVT089 and WD112 were used to amplify alinker fragment. Both PCR™ products were digested with TaqI and ligatedto each other. The ligation product was gel purified and PCR™ amplifiedusing primer pair MVT089 and FW006. The amplified product was digestedwith MluI and SpeI and ligated into MluI and SpeI digested and purifiedpEG1701 vector DNA. These plasmids as a group are designatedpEG1701.lβ6,7.

5.22.6 pEG1701.lβ10,11

Plasmids containing mutated cry3Bb sequences at nucleotides 1450-1467,encoding structural region lβ10,11 of Cry3Bb, were constructed byreplacing the SpeI-PstI fragment of pEG1701 with isolated SpeI- andPstI-digested PCR™ product. The PCR™ product was generated byquasi-random PCR™ mutagenesis where mutagenic primer MVT105 was pairedwith primer MVT070. Primer pair MVT092 and MVT083 were used to generatea linker fragment. (MVT083 is a mutagenic oligo designed for anotherregion. The sequence changes introduced by MVT083 are removed followingrestriction digestion and do not impact the alteration of cry3Bb in thelβ10,11 region.) Both PCR™ products were digested with BsaI, ligatedtogether, and the ligation product PCR™ amplified with primer pairMVT083 and MVT070. The resulting PCR™ product was digested with SpeI andPstI, and gel purified. These plasmids as a group are designatedpEG1701.lβ10,11.

5.23 Example 23 DNA Shuffling

DNA-shuffling, as described by Stemmer (1994), was used to combineindividual alterations in the cry3Bb gene.

5.23.1 pEG1084, pEG1085, pEG1086 and pEG1087

pEG1084, pEG1085, pEG1086, and pEG1087, contained in EG11081, EG11082,EG11083, and EG11084, respectively, were recovered from DNA-shuffling.Briefly, PflMI DNA fragments were generated using primer set A and B andeach of the plasmids pEG1707, pEG1714, pEG1715, pEG1716, pEG1041,pEG1046, pEG1047, and pEG1054 as DNA templates. The resulting DNAfragments were pooled in equal-molar amounts and digested with DNaseIand 50-100 bp DNA fragments were recovered from an agarose gel by threesuccessive freeze-thaw cycles: three min in a dry-ice ethanol bathfollowed by complete thawing at 50° C. The recovered DNA fragments wereassembled by primerless-PCR™ and PCR™-amplified using the primer set Aand B as described by Stemmer (1994). The final PCR™-amplified DNAfragments were cut with PflMI and used to replace the correspondingcry3Bb PflMI DNA fragment in pEG1701.

5.24 Example 24 Recombinant DNA Techniques

Standard recombinant DNA procedures were performed essentially asdescribed by Sambrook et al. (1989).

5.24.1 pEG1717

pEG1717, contained in EG11231, was constructed by replacing the smallBglII fragment of pEG1710 with the small BglII fragment from pEG1714.

5.24.2 pEG1721

pEG1721, contained in EG11235, was constructed by replacing the smallBglII fragment from pEG1710 with the small BglII fragment from pEG1087.

5.24.3 pEG1063

pEG1062, contained in EG11057, was constructed by replacing the NcoI DNAfragment containing ori 43 from pEG1054 with the isolated NcoI DNAfragment containing ori 43 and the alterations in cry3Bb from pEG1046.

5.24.4 pEG1063

pEG1063, contained in EG11058, was constructed by replacing the NcoI DNAfragment containing ori 43 from pEG1054 with the isolated NcoI DNAfragment containing ori 43 and the alterations in cry3Bb from pEG1707.

5.24.5 pEG1095

pEG1095, contained in EG11095, was constructed by replacing theMluI-SpeI DNA fragment in pEG1701 with the corresponding MluI-SpeI DNAfragment from pEG1086.

5.25 Example 25 Primers Utilized in Constructing Cry3Bb* Variants

Shown below are the primers used for site-directed mutagenesis, triplexPCR™ and quasi-random PCR™ to prepare the cry3Bb* variants as describedabove. Primers were obtained from Ransom Hill Bioscience, Inc. (Ramona,Calif.) and Integrated DNA Technologies, Inc. (Coralville, Iowa). Thespecific composition of the primers containing particular degeneraciesat one or more residues is given in Section 5.30, Example 30.

5.25.1 PRIMER FW001: 5′-AGACAACTCTACAGTAAAAGATG-3′ (SEQ ID NO:71) 5.25.2PRIMER FW006: 5′-GGTAATTGGTCAATAGAATC-3′ (SEQ ID NO:72) 5.25.3 PRIMERMVT095: 5′-CAGAAGATGTTGCTGAATTCNNNCATAGACAATTAAAAC-3′ (SEQ ID NO:73)5.25.4 PRIMER MVT097: 5′-GATGTTGCTGAATTCTATNNNAGACAATTAAAAC-3′ (SEQ IDNO:74) 5.25.5 PRIMER MVT091: 5′-CCCATTTTATGATATTBDNTTATACTCAAAAGG-3′(SEQ ID NO:75) 5.25.6 PRIMER MVT075:5′-AGCTATGCTGGTCTCGGAAGAAAEFNFFNFINJFJFJNFINJFJAAAAGAAGCCAAGATCGAAT-3′(SEQ ID NO:76) 5.25.7 PRIMER MVT076:5′-GGTCACCTAGGTCTCTCTTCCAGGAATTTAACGCATTAAC-3′ (SEQ ID NO:77) 5.25.8PRIMER MVT111:5′-AGCTATGCTGGTCTCCCATTTJEHIEJEJJEIIKRRJEHEIJEENIIIGTTAAAACAGAACTAAC-3′(SEQ ID NO:78) 5.25.9 PRIMER MVT094:5′-ATCCAGTGGGGTCTCAAATGGGAAAAGTACAATTAG-3′ (SEQ ID NO:79) 5.25.10 PRIMERMVT103:5′-CATTTTTACGGATCCAATTTTTJFFFJNEEJEFNFJNFEILEIJEOGGACCAACTTTTTTGAG-3′(SEQ ID NO:80) 5.25.11 PRIMER MYT081:5′-GAATTTCATACGCGTCTTCAACCTGGTJEHJJJIINMEEIEJTCTTTCAATTATTGGTCTGG-3′(SEQ ID NO:81) 5.25.12 PRIMER MVT085:5′-AAAAGTTTATCGAACTATAGCTAATACAGACGTAGCGGCTJQQFFNEEJIIIJEEIGTATATTTAGGTGTTACG-3′(SEQ ID NO:82) 5.25.13 PRIMER A 3B2PFLM1: 5′-GGAGTTCCATTTGCTGGGGC-3′(SEQ ID NO:83) 5.25.14 PRIMER B 3B2PFLM2: 5′-ATCTCCATAAAATGGGG-3′ (SEQID NO:84) 5.25.15 PRIMER C 3B2165DG:5′-GCGAAGTAAAAGAAGCCAAGGTCGAATAAGGG-3′ (SEQ ID NO:85) 5.25.16 PRIMER D3B2160SKRD: 5′-CCTTTAAGTTTGCGAAATCCACACAGCCAAGGTCGAATAAGGG-3′ (SEQ IDNO:86) 5.25.17 PRIMER E 3B2290VP:5′-CCCATTTTATGATGTTCGGTTATACCCAAAAGGGG-3′ (SEQ ID NO:87) 5.25.18 PRIMERF 3B2EDA104: 5′-GGCCAAGTGAAGACCCATGGAAGGC-3′ (SEQ ID NO:88) 5.25.19PRIMER G 3B2KG189: 5′-GCAGTTTCCGGATTCGAAGTGC-3′ (SEQ ID NO:89) 5.25.20PRIMER WD112: 5′-CCGCTACGTCTGTATTA-3′ (SEQ ID NO:90) 5.25.21 PRIMERWD115: 5′-ATAATGGAAGCACCTGA-3′ (SEQ ID NO:91) 5.25.22 PRIMER MVT105:5′-AGCTATGCTGGTCTCTTCTTAEJIFEIIEFFIJFIJIINACAATTCCATTTTTTACTTGG-3′ (SEQID NO:92) 5.25.23 PRIMER MVT092:5′-ATCCAGTTGGGTCTCTAAGAAACAAACCGCGTAATTAAGC-3′ (SEQ ID NO:93) 5.25.24PRIMER MVT070: 5′-CCTCAAGGGTTATAACATCC-3′ (SEQ ID NO:94) 5.25.25 PRIMERMVT083: 5′-GTACAAAAGCTAAGCTTTIEJIINPEEMEEIJNJESCGAACTATAGCTAATACAG-3′(SEQ ID NO:95)

5.26 Example 26 Sequence Analysis of Altered Cry3Bb Genes

E. coli DH5α™ (GIBCO BRL, Gaithersburg, Md.), JM110 and Sure™(Stratagene, La Jolla, Calif.) cells were sometimes used amplify plasmidDNA for sequencing. Plasmids were transformed into these cells using themanufacturers' procedures. DNA was sequenced using the Sequenase® 2.0DNA sequencing kit purchased from U.S. Biochemical Corporation(Cleveland, Ohio). The plasmids described in Section 6, their respectivedivergence from WT cry3Bb sequence, the resulting amino acid changes andthe protein structure site of the changes are listed in Table 11.

TABLE 11 DNA SEQUENCE CHANGES OF CRY3BB* GENES AND RESULTING AMINO ACIDSUBSTITUTIONS OF THE CRY3BB* PROTEINS Structural Site of Plasmid cry3Bb*DNA Sequence Cry3Bb* Amino Acid Sequence Alteration pEG1707 A460T,C461T, A462T, C464A, T465C, T466C, T467A, T154F, P155H, L156H, L158R1α3, 4 A468T, A469T, G470C, T472C, T473G, G474T, A477T, A478T, G479CpEG1708 T687C, T688C, A689T, C691A, A692G Y230L, H231S α6 pEG1709 T667C,T687C, T688A, A689G, C691A, A692G S223P, Y230S α6 pEG1710 T687C, A692GH231R α6 pEG1711 T687C, C691A H231N, T241S α6 pEG1712 T687C, C691A,A692C, T693C H231T α6 pEG1713 C868A, G869A, G870T R290N 1α7, β1 pEG1714C932T, A938C, T942G, G949A, T954C S311L, N313T, E317K 1β1, α8 pEG1715T931A, A933C, T942A, T945A, G949A, A953G, S311T, E317K, Y318C 1β1, α8T954C pEG1716 T931G, A933C, C934G, T945G, C946T, A947G, S311A, L312V,Q316W 1β1, α8 G951A, T954C pEG1717 T687C, A692G, C932T, A938C, T942G,G949A, H231R, S311L, N313T, E317K α6, 1β1, α8 T954C pEG1718 T931A,A933G, T935C, T936A, A938C, T939C, S311T, L312P, N313T, E317N 1β1, α8T942C, T945A, G951T, T954C pEG1719 T931G, A933C, T936G, T942C, C943T,T945A, S311A, Q316D 1β1, α8 C946G, G948C, T954C pEG1720 T861C, T866C,C868A, T871C, T872G, A875T, I289T, L291R, Y292F, S293R 1α7, β1 T877A,C878G, A882G pEG1721 T687C, A692G, C932T H231R, S311L α6, 1β1, α8pEG1722 T931A, C932T, A933C, T936C, T942G, T945A, T954C S311I 1β1, α8pEG1723 T931A, C932T, A933C, T936C, A937G, A938T, S311I, N313H 1β1, α8C941A, T942C, T945A, C946A, A947T, A950T, T954C pEG1724 A933C, T936C,A937G, A938T, C941A, T942C, N313V, T314N, Q316M, E317V 1β1, α8 T945A,C946A, A947T, A950T, T954C pEG1725 A933T, A938G, T939G, T942A, T944C,T945A, N313R, L315P, Q316L, E317A 1β1, α8 A947T, G948T, A950C, T954CpEG1726 A860T, T861C, G862A, C868T, G869T, T871C, Y287F, D288N, R290L1α7, β1 A873T, T877A, C878G, A879T pEG1727 C868G, G869T R290V 1α7, β1pEG1041 A494G D165G α4 pEG1046 G479A, A481C, A482C, A484C, G485A, S160N,K161P, R162H, D165G α4 A486C, A494G pEG1047 A865G, T877C I289V, S293P1α7, β1 pEG1052 G479A, A481C, A482C, A484C, G485A, A486C, S160N, K161P,R162H, D165G, α4, 1α7, β1 A494G, A865G, T877C I289V, S293P pEG1054T309A, Δ310, Δ311, Δ312 D103E, ΔA104 1α2a, 2b pEG1057 A565G, A566G K189G1α4, 5 pEG1062 T309A, Δ310, Δ311, Δ312, G479A, A481C, A482C, D103E,ΔA104, S160N, K161P, 1α2a, 2b α4 A484C, G485A, A486C, A494G R162H, D165GpEG1063 T309A, Δ310, Δ311, Δ312, A460T, C461T, A462T, D103E, ΔA104,T154F, P155H, 1α2a, 2b 1α3, 4 C464A, T465C, T466C, T467A, A468T, A469T,L156H, L158R G470C, T472C, T473G, G474T, A477T, A478T, G479C pEG1084A494G, T931A, A933C, T942A, T945A, G949A, D165G, S311T, E317K α4, 1β1,α8 T954C pEG1085 A494G, A865G, T877C, T914C, T931G, A933C, D165G, I289V,S293P, F305S, α4, 1α7, β1, β1, 1β1, α8 C934G, T945G, C946T, A947G,G951A, T954C, S311A, L312V, Q316W, Q348R, β2, β3b A1043G, T1094C V365ApEG1086 A865G, T877C, A1043G I289V, S293P, Q348R 1α7, β1, β2 pEG1087A494G, C932T D165G, S311L α4, 1β1, α8 pEG1095 A1043G Q348R β2

5.27 Example 27 Expression of Cry3Bb* Proteins

5.27.1 Culture Conditions

LB agar was prepared using a standard formula (Maniatis et al., 1982).Starch agar was obtained from Difco Laboratories (Detroit, Mich.) andsupplemented with an additional 5 g/l of agar. C2 liquid medium isdescribed by Donovan et al. (1988). C2 medium was sometimes preparedwithout the phosphate buffer (C2-P). All cultures were incubated at 25°C. to 30° C.; liquid cultures were also shaken at 250 rpm, untilsporulation and lysis had occurred.

5.27.2 Transformation Conditions

pEG1701 and derivatives thereof were introduced into acrystalliferiousB. thuringiensis var. kurstaki EG7566 (Baum, 1994) or EG10368 (U.S. Pat.No. 5,322,687) by the electroporation method of Macaluso and Mettus(1991). In some cases, the method was modified as follows to maximizethe number of transformants. The recipient B. thuringiensis strain wasinoculated from overnight growth at 30° C. on LB agar into brain heartinfusion plus 0.5% glycerol, grown to an optical density ofapproximately 0.5 at 600 nm, chilled on ice for 10 min, washed 2× withEB and resuspended in a 1/50 volume of EB. Transformed cells wereselected on LB agar or starch agar plus 5 μg/ml chloramphenicol. Visualscreening of colonies was used to identify transformants producingcrystalline protein; those colonies were generally more opaque thancolonies that did not produce crystalline protein.

5.27.3 Strain and Protein Designations

A transformant containing an altered cry3Bb* gene encoding an alteredCry3Bb* protein is designated by an “EG” number, e.g., EG11231. Thealtered Cry3Bb* protein is designated Cry3Bb followed by the strainnumber, e.g., Cry3Bb.11231. Collections of proteins with alterations ata structural site are designated Cry3Bb followed by the structural site,e.g., Cry3Bb.lβ2,3. Table 12 lists the plasmids pertinent to thisinvention, the new B. thuringiensis strains containing the plasmids, theacrystalliferous B. thuringiensis recipient strain used, and theproteins produced by the new strains.

5.28 Example 28 Generation and Characterization of Cry3Bb-60

5.28.1 Generation of Cry3Bb-60

Cry3Bb-producing strain EG7231 (U.S. Pat. No. 5,187,091) was grown in C2medium plus 3 mg/ml chloramphenicol. Following sporulation and lysis,the culture was washed with water and Cry3Bb protein purified by theNaBr solubilization and recrystallization method of Cody et al. (1992).Protein concentration was determined by BCA Protein Assay (Pierce,Rockford, Ill.). Recrystallized protein was solubilized in 10 ml of 50mM KOH per 100 mg of Cry3Bb protein and buffered to pH 9.0 with 100 mMCAPS (3-[cyclohexylamino]-1-propanesulfonic acid), pH 9.0. The solubletoxin was treated with trypsin at a weight ratio of 50 mg toxin to 1 mgtrypsin for 20 min to overnight at room temperature. Trypsin cleavesproteins on the carboxyl side of available arginine and lysine residues.For 8-dose bioassay, the solubilization conditions were altered slightlyto increase the concentration of protein: 50 mM KOH was added dropwiseto 2.7 ml of a 12.77 mg/ml suspension of purified Cry3Bb* until crystalsolubilization occurred. The volume was then adjusted to 7 ml with 100mM CAPS, pH 9.0.

TABLE 12 PLASMIDS CARRYING ALTERED CRY3BB* GENES TRANSFORMED INTO B.THURINGIENSIS FOR EXPRESSION OF ALTERED CRY3BB* PROTEINS PlasmidDesignation New BT Strain Expressed Protein pEG1701 EG11204 WT Cry3BbpEG1701 EG11037 WT Cry3Bb pEG1707 EG11221 Cry3Bb.11221 pEG1708 EG11222Cry3Bb.11222 pEG1709 EG11223 Cry3Bb.11223 pEG1710 EG11224 Cry3Bb.11224pEG1711 EG11225 Cry3Bb.11225 pEG1712 EG11226 Cry3Bb.11226 pEG1713EG11227 Cry3Bb.11227 pEG1714 EG11228 Cry3Bb.11228 pEG1715 EG11229Cry3Bb.11229 pEG1716 EG11230 Cry3Bb.11230 pEG1717 EG11231 Cry3Bb.11231pEG1718 EG11232 Cry3Bb.11232 pEG1719 EG11233 Cry3Bb.11233 pEG1720EG11234 Cry3Bb.11234 pEG1721 EG11235 Cry3Bb.11235 pEG1722 EG11236Cry3Bb.11236 pEG1723 EG11237 Cry3Bb.11237 pEG1724 EG11238 Cry3Bb.11238pEG1725 EG11239 Cry3Bb.11239 pEG1726 EG11241 Cry3Bb.11241 pEG1727EG11242 Cry3Bb.11242 pEG1041 EG11032 Cry3Bb.11032 pEG1046 EG11035Cry3Bb.11035 pEG1047 EG11036 Cry3Bb.11036 pEG1052 EG11046 Cry3Bb.11046pEG1054 EG11048 Cry3Bb.11048 pEG1057 EG11051 Cry3Bb.11051 pEG1062EG11057 Cry3Bb.11057 pEG1063 EG11058 Cry3Bb.11058 pEG1084 EG11081Cry3Bb.11081 pEG1085 EG11082 Cry3Bb.11082 pEG1086 EG11083 Cry3Bb.11083pEG1087 EG11084 Cry3Bb.11084 pEG1095 EG11095 Cry3Bb.11095 pEG1098EG11098 Cry3Bb.11098 pEG1701.1β2, 3 collection of unnamed strainsCry3Bb.1β2, 3 pEG1701.1β6, 7 collection of unnamed strains Cry3Bb.1β6, 7pEG1701.1β10, 11 collection of unnamed strains Cry3Bb.1β10, 115.28.2 Determination of Molecular Weight of Cry3Bb-60

The molecular weight of the predominant trypsin digestion fragment ofCry3Bb was determined to be 60 kDa by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis using commercial molecular weightmarkers. This digestion fragment is designated Cry3Bb-60. No furtherdigestion of the 60 kDa cleavage product was observed.

5.28.3 Determination of NH₂-Terminus of Cry3Bb-60

To determine the NH₂-terminal sequence of Cry3Bb-60, the trypsin digestwas fractionated by SDS-PAGE and transferred to Immobilon™-P membrane(Millipore Corporation, Bedford, Mass.) following standard westernblotting procedures. After transfer, the membrane was rinsed twice withwater then stained with 0.025% Coomassie Brilliant Blue R-250 plus 40%methanol for 5 min, destained with 50% methanol and rinsed in water. TheCry3Bb.60 band was excised with a razor blade. NH₂-terminal sequencingwas performed at the Tufts Medical School, Department of Physiology(Boston, Mass.) using standard automated Edman degradation procedures.The NH₂-terminal amino acid sequence was determined to be SKRSQDR (SEQID NO:96), corresponding to amino acids 160-166 of Cry3Bb. Trypsindigestion occurred on the carboxyl side of amino acid R159 resulting inthe removal of helices 1-3.

5.29 Example 29 Bioactivity of Cry3Bb* Proteins

5.29.1 Culture Conditions and Protein Concentration Determination

Cultures for 1-dose bioassays were grown in C2-P plus 5 μg/mlchloramphenicol (C2-P/cm5) then diluted with 3 volumes of 0.005% TritonX-100®. The protein concentrations of these cultures were notdetermined. Cultures for 8-dose bioassays were grown in C2/cm5, washed1-2 times with 1-2 volumes of sterile water and resuspended in 1/10volume of sterile 0.005% Triton X-100®. The toxin protein concentrationof each concentrate was determined as described by Brussock and Currier(1990), omitting the treatment with 3 M HEPES. The protein concentrationwas adjusted to 3.2 mg/ml in 0.005% Triton X-100® for the top dose ofthe assay. Cry3Bb.60 was produced and quantified for 8-dose assay asdescribed in Section 9.1.

5.29.2 Insect Bioassays

Diabrotica undecimpunctata howardi Barber (southern corn rootworm orSCRW) and Diabrotica virgifera virgifiera LeConte (western corn rootwormor WCRW) larvae were reared as described by Slaney et al. (1992).Eight-dose assays and probit analyses were performed as described bySlaney et al. (1992). Thirty-two larvae were tested per dose at 50 μl ofsample per well of diet (surface area of 175 mm²). Positive controlswere WT Cry3Bb-producing strains EG11037 or EG11204. All bioassays wereperformed using 128-well trays containing approximately 1 ml of diet perwell with perforated mylar sheet covers (C-D International Inc., Pitman,N.J.). One-dose assays were performed essentially the same except only 1dose was tested per strain. All assay were replicated at least twice.

5.29.3 Insect Bioassay Results: 1-Dose Assays Against SCRW

Results from 1-dose assays are expressed as the relative mortality (RM)of the experimental strain compared to WT (% mortality of experimentalculture divided by % mortality of WT culture). Altered and improvedCry3Bb proteins derived from plasmids constructed using PCR™ methodsintroducing random or semi-random changes into the cry3Bb gene sequencewere distinguished from other altered but not improved Cry3Bb proteinsby replicated, 1-dose assay against SCRW larvae. Those proteins showingincreased activity (defined as RM≧1.5) compared to WT Cry3Bb or, in thecase of proteins with combinations of altered sites, compared to a“parental” altered Cry3Bb protein were further characterized by 8-doseassay. The overall RM “pattern” produced by 1-dose assay results from acollection of proteins carrying random or semi-random alterations withina single structural region, e.g., in lβ2,3, can be used to determine ifthat structural region is important for bioactivity. Retention of WTlevels of activity (RM≈1) indicate changes are tolerated in that region.Overall loss of activity (RM<1) distinguishes the region as importantfor bioactivity.

5.29.4 Cry3Bb.lβ2,3: Results of 1-Dose Bioassays Against SCRW

Cry3Bb.lβ2.3 protein are a collection of proteins altered in the lβ2,3region of Cry3Bb (see Section 5.3.4). Typical results of 1-dose assaysof these altered proteins are shown in FIG. 12. The RM values forCry3Bb.lβ2,3 proteins are less than 1, with a few exceptions of valuesclose to 1, indicating that this region is important for toxicity.

5.29.5 Cry3Bb.lβ6,7: Results of 1-Dose Bioassays Against SCRW

Cry3Bb.lβ6,7 proteins are a collection of proteins altered in the lβ6,7region of Cry3Bb (see Section 5.3.5). Typical results of 1-dose assaysof these altered proteins are shown in FIG. 13. With a few exceptions ofvalues close to 1, the RM values for Cry3Bb.lβ6,7 proteins are less than1, indicating that this region is important for toxicity.

5.29.6 Cry3Bb.lβP10,11: Results of 1-Dose Bioassays Against SCRW

Cry3Bb.lβ10,11 proteins are a collection of proteins altered in thelβ10,11 region of Cry3Bb (see Section 5.3.6). Typical results of 1-doseassays of these altered proteins are shown in FIG. 14. With a fewexceptions of values close to 1, the RM values for Cry3Bb.lβ10,11proteins are less than 1, indicating that this region is important forbioactivity.

5.29.7 Insect Bioassay Results: Results of 8-Dose Assays Against SCRW

Results from 8-dose assays are expressed as an LC₅₀ value (proteinconcentration giving 50% mortality) with 95% confidence intervals. TheLC₅₀ values with 95% confidence intervals of altered Cry3Bb proteinsshowing improved activities against SCRW larvae and LC₅₀ values of theWT Cry3Bb control determined at the same time are listed in Table 13along with the fold increase over WT activity for each improved protein.

TABLE 13 DESIGNED CRY3BB PROTEINS WERE TESTED AGAINST SCRW LARVAE INREPLICATED, 8-DOSE ASSAYS TO DETERMINE THE LC₅₀ VALUES LC₅₀ μg/well (95%C.I.) WT Cry3Bb Fold Increase Improved Protein Improved Protein ControlOver WT Activity Cry3Bb.60  6.7 (5.3–8.4) 24.1 (15–39) 3.6× Cry3Bb.11221 3.2 (2.5–4) 20.5 (14.5–29) 6.4× Cry3Bb.11222  7.3 (6–9) 29.4 (23–37)4.0× Cry3Bb.11223 10.5 (9–12) 29.4 (23–37) 2.8× Cry3Bb.11224  6.5(5.1–8.2) 32.5 (25–43) 5.0× Cry3Bb.11225 13.7 (11–16.8) 49.5 (39–65)3.6× Cry3Bb.11226 16.7 (10.6–24.2) 49.5 (39–65) 3.0× Cry3Bb.11227 11.1(9.1–13.5) 21.3 (16–28) 1.9× Cry3Bb.11228  8.0 (6.6–9.8) 32.9 (25–45)4.1× Cry3Bb.11229  7.2 (5.8–8.8) 18.2 (15–22) 2.5× Cry3Bb.11230  7.0(5.8–8.6) 32.9 (25–45) 4.7× Cry3Bb.11231  3.3 (3.0–3.7) 26.1 (22–31)7.9× Cry3Bb.11232  6.4 (5.4–7.7) 32.9 (25–45) 5.1× Cry3Bb.11233 15.7(12–20) 32.9 (25–45) 2.2× Cry3Bb.11234   7 (6–9)   29 (22–39) 4.1×Cry3Bb.11235  4.2 (3.6–4.9) 13.3 (10–17) 3.2× Cry3Bb.11236 11.6 (9–15)36.4 (27–49) 3.1× Cry3Bb.11237  6.8 (4–11) 36.4 (27–49) 5.4×Cry3Bb.11238 13.9 (11–17) 36.4 (27–49) 2.6× Cry3Bb.11239 13.0 (10–16)36.4 (27–49) 2.8× Cry3Bb.11241   11 (7–16)   29 (22–39) 2.6×Cry3Bb.11242 11.9 (9.2–16)   30 (23–38) 2.5× Cry3Bb.11032  4.2 (3.6–4.9)13.3 (10–17) 3.1× Cry3Bb.11035 10.3 (8–13) 27.9 (23–34) 2.7×Cry3Bb.11036  6.5 (5.1–7.9) 27.9 (23–34) 4.3× Cry3Bb.11046 12.1 (8–19)31.2 (25–39) 2.6× Cry3Bb.11048  8.3 (6–11) 35.4 (24–53) 4.3×Cry3Bb.11051 11.8 (8–16) 35.4 (24–53) 3.0× Cry3Bb.11057  8.8 (7–11) 29.5(24–36) 3.4× Cry3Bb.11058  9.6 (6–14) 33.4 (27–43) 3.5× Cry3Bb.11081 8.5 (7–11) 51.5 (37–79) 6.1× Cry3Bb.11082 10.6 (8–13) 51.5 (37–79) 4.9×Cry3Bb.11083  7.0 (5–10) 51.5 (37–79) 7.4× Cry3Bb.11084  7.2 (4–12) 51.5(37–79) 7.2× Cry3Bb.11095 11.1 (9–14) 51.5 (37–79) 4.6× Cry3Bb.110985.29.8 Insect Bioassay Results: 8-Dose Assays Against WCRW

WCRW larvae are delicate and difficult to work with. Therefore, onlysome of the designed Cry3Bb showing improved activity against SCRWlarvae were also tested against WCRW larvae in 8-dose assays. The LC₅₀determinations for the designed Cry3Bb proteins are shown in Table 14along with the LC₅₀ values of the WT Cry3Bb control determined at thesame time.

TABLE 14 CRY3BB* PROTEINS SHOWING IMPROVED ACTIVITY AGAINST SCRW LARVAEALSO SHOW IMPROVED ACTIVITY AGAINST WCRW LARVAE LC₅₀ μg/well (95% C.I.)WT Cry3Bb Fold Increase Improved Protein Improved Protein Control OverWT Activity EG11083  6.3 (4.7–8.2) 63.5 (46–91) 10.1× EG11230 24.2(13–40)  4.5 (2.1–7.4) 5.4× EG11231 32.2 (14–67)  2.5 (1.7–3.6) 12.9×

5.30 Example 30 Channel Activity

Ion channels produced by Cry3Bb and some of its derivatives weremeasured by the methods described by Slatin et al. (1990). In someinstances, lipid bilayers were prepared from a mixture of 4:1phophatidylethanolamine (PE):phosphatidylcholine (PC). Toxin protein wassolubilized from washed, C2 medium, B. thuringiensis cultures with 12 mMKOH. Following centrifugation to remove spores and other debris, 10 μgof soluble toxin protein was added to the cis compartment (4.5 mlvolume) of the membrane chamber. Protein concentration was determinedusing the BCA Protein Assay (Pierce).

5.30.1 Channel Activity of WT Cry3Bb.

Upon exposure to black lipid membranes, Cry3Bb forms ion channels withvarious conductance states. The channels formed by Cry3Bb are rarelydiscrete channels with well resolved open and closed states and usuallyrequire incubation of the toxin with the membrane for 30-45 min beforeany channel-like events are observed. After formation of the initialconductances, the size increases from approximately 200 pS to over10,000 pS over 2-3 h. Only the small conductances (≦200 pS) are voltagedependent. Over 200 pS, the conductances are completely symmetric.Cry3Bb channels also exhibit β-mercaptoethanol-dependent activation,growing from small channel conductances of ˜200 pS to several thousandpS within 2 min of the addition of β-mercaptoethanol to the ciscompartment of the membrane chamber.

5.30.2 Cry3Bb.11032

The channel activity of Cry3Bb.11032 is much like WT Cry3Bb when thesolubilized toxin protein is added to the cis compartment of themembrane chamber. However, when this protein is artificiallyincorporated into the membrane by forming or “painting” the membrane inthe presence of the Cry3Bb.11032 protein, a 16-fold increase in theinitial channel conductances is observed (˜4000 pS). This phenomenon isnot observed with WT Cry3Bb.

5.30.3 Cry3Bb.11035

Upon exposure to artificial membranes, the Cry3Bb.11035 proteinspontaneously forms channels that grow to large conductances within arelatively short time span (˜5 min). Conductance values ranges from3000-6000 pS and, like WT Cry3Bb, are voltage dependent at lowconductance values.

5.30.4 Cry3Bb.11048

The Cry3Bb.11048 protein is quite different than WT Cry3Bb in that itappears not to form channels at all, but, rather, forms symmetricalpores with respect to voltage. Once the pore is formed, it remains openand allows a steady conductance ranging from 25 to 130 pS.

5.30.5 Cry3Bb.11224 and Cry3Bb.11226

The metal binding site of WT Cry3Bb formed by H231 in the dimerstructure was removed in proteins Cry3Bb.11224 and Cry3Bb.11226. Theconductances formed by both designed proteins are identical to that ofWT Cry3Bb with the exception that neither of the designed proteinsexhibits β-mercaptoethanol-dependent activation.

5.30.6 Cry3Bb.11221

Cry3Bb.11221 protein has been observed to immediately form smallchannels of 100-200 pS with limited voltage dependence. Some higherconductances were observed at the negative potential. In other studiesthe onset of activity was delayed by 27 min, which is more typical forWT Cry3Bb. Unlike WT Cry3Bb, however, Cry3Bb.11221 forms well resolved,600 pS channels with long open states. The protein eventually reachesconductances of 7000 pS.

5.30.7 Cry3Bb.11242

Cry3Bb.11242 protein forms small conductances immediately upon exposureto an artificial membrane. The conductances grow steadily and rapidly to6000 pS in approximately 3 min. Some voltage dependence was noted with apreference for a negative imposed voltage.

5.30.8 Cry3Bb.11230

Unlike WT Cry3Bb, Cry3Bb.11230 forms well resolved channels with longopen states that do not continue to grow in conductance with time. Themaximum observed channel conductances reached 3000 pS. FIG. 15illustrates the difference between the channels formed by Cry3Bb andCry3Bb.11230.

5.30.9 Cry3Bb.60

Cry3Bb.60 forms well resolved ion channels within 20 min of exposure toan artificial membrane. These channels grow in conductance and frequencywith time. The behavior of Cry3Bb.60 in a planar lipid bilayer differsfrom Cry3Bb in two significant ways. The conductances created byCry3Bb.60 form more quickly than Cry3Bb and, unlike Cry3Bb, theconductances are stable, having well resolved open and closed statesdefinitive of stable ion channels (FIG. 16).

5.31 Example 31 Primer Compositions

TABLE 15 SEQ ID NO:83 % of Nucleotide in mixture Code A T G C N 25 25 2525

TABLE 16 SEQ ID NO:84 % of Nucleotide in mixture Code A T G C N 25 25 2525

TABLE 17 SEQ ID NO:85 % of Nucleotide in mixture Code A T G C B 16 16 5216 D 70 10 10 10 N 25 25 25 25

TABLE 18 SEQ ID NO:86 % of Nucleotide in mixture Code A T G C E 82 6 6 6F 6 6 6 82 J 6 82 6 6 I 6 6 82 6 N 25 25 25 25

TABLE 19 SEQ ID NO:88 % of Nucleotide in mixture Code A T G C J 6 82 6 6E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 K 15 15 15 55 R 15 55 15 15

TABLE 20 SEQ ID NO:90 % of Nucleotide in mixture Code A T G C J 6 82 6 6F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6 L 8 1 83 8 O 1 1 1 97

TABLE 21 SEQ ID NO:91 % of Nucleotide in mixture Code A T G C J 6 82 6 6E 82 6 6 6 H 1 1 1 97 I 6 6 82 6 N 25 25 25 25 M 82 2 8 8

TABLE 22 SEQ ID NO:92 % of Nucleotide in mixture Code A T G C J 6 82 6 6Q 0 9 82 9 F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6

TABLE 23 SEQ ID NO:92 % of Nucleotide in mixture Code A T G C J 6 82 6 6F 6 6 6 82 N 25 25 25 25 E 82 6 6 6 I 6 6 82 6

TABLE 24 SEQ ID NO:95 % of Nucleotide in mixture Code A T G C J 6 82 6 6N 25 25 25 25 E 82 6 6 6 I 6 6 82 6 M 82 2 8 8 P 8 2 8 82 S 1 97 1 1

5.32 Example 32 Atomic Coordinates for Cry3Bb

The atomic coordinates of the Cry3Bb protein are given in the Appendixincluded in Section 9.1

5.33 Example 33 Atomic Coordinates for Cry3A

The atomic coordinates of the Cry3A protein are given in the Appendixincluded in Section 9.2

5.34 Example 34 Modification of Cry Genes for Expression in Plants

Wild-type cry genes are known to be expressed poorly in plants as a fulllength gene or as a truncated gene. Typically, the G+C content of a crygene is low (37%) and often contains many A+T rich regions, potentialpolyadenylation sites and numerous ATTTA sequences. Table 25 shows alist of potential polyadenylation sequences which should be avoided whenpreparing the “plantized” gene construct.

TABLE 25 LIST OF SEQUENCES OF THE POTENTIAL POLYADENYLATION SIGNALSAATAAA* AAGCAT AATAAT* ATTAAT AACCAA ATACAT ATATAA AAAATA AATCAAATTAAA** ATACTA AATTAA** ATAAAA AATACA** ATGAAA CATAAA** *indicates apotential major plant polyadenylation site. **indicates a potentialminor animal polyadenylation site. All others are potential minor plantpolyadenylation sites.

The regions for mutagenesis may be selected in the following manner. Allregions of the DNA sequence of the cry gene are identified whichcontained five or more consecutive base pairs which were A or T. Thesewere ranked in terms of length and highest percentage of A+T in thesurrounding sequence over a 20-30 base pair region. The DNA is analysedfor regions which might contain polyadenylation sites or ATTTAsequences. Oligonucleotides are then designed which maximize theelimination of A+T consecutive regions which contained one or morepolyadenylation sites or ATTTA sequences. Two potential plantpolyadenylation sites have been shown to be more critical based onpublished reports. Codons are selected which increase G+C content, butdo not generate restriction sites for enzymes useful for cloning andassembly of the modified gene (e.g, BamHI, BglII, SacI, NcoI, EcoRV,etc.). Likewise condons are avoided which contain the doublets TA or GCwhich have been reported to be infrequently-found codons in plants.

Although the CaMV35S promoter is generally a high level constitutivepromoter in most plant tissues, the expression level of genes driven theCaMV35S promoter is low in floral tissue relative to the levels seen inleaf tissue. Because the economically important targets damaged by someinsects are the floral parts or derived from floral parts (e.g., cottonsquares and bolls, tobacco buds, tomato buds and fruit), it is oftenadvantageous to increase the expression of crystal proteins in thesetissues over that obtained with the CaMV35S promotor.

The 35S promoter of Figwort Mosaic Virus (FMV) is analogous to theCaMV35S promoter. This promoter has been isolated and engineered into aplant transformation vector. Relative to the CaMV promoter, the FMV 35Spromoter is highly expressed in the floral tissue, while still providingsimilar high levels of gene expression in other tissues such as leaf. Aplant transformation vector, may be constructed in which the full lengthsynthetic cry gene is driven by the FMV 35S promoter. Tobacco plants maybe transformed with the vector and compared for expression of thecrystal protein by Western blot or ELISA immunoassay in leaf and floraltissue. The FMV promoter has been used to produce relatively high levelsof crystal protein in floral tissue compared to the CaMV promoter.

5.35 Example 35 Expression of Synthetic Cry Genes with ssRUBISCOPromoters and Chloroplast Transit Peptides

The genes in plants encoding the small subunit of RUBISCO (SSU) areoften highly expressed, light regulated and sometimes show tissuespecificity. These expression properties are largely due to the promotersequences of these genes. It has been possible to use SSU promoters toexpress heterologous genes in transformed plants. Typically a plant willcontain multiple SSU genes, and the expression levels and tissuespecificity of different SSU genes will be different. The SSU proteinsare encoded in the nucleus and synthesized in the cytoplasm asprecursors that contain an N-terminal extension known as the chloroplasttransit peptide (CTP). The CTP directs the precursor to the chloroplastand promotes the uptake of the SSU protein into the chloroplast. In thisprocess, the CTP is cleaved from the SSU protein. These CTP sequenceshave been used to direct heterologous proteins into chloroplasts oftransformed plants.

The SSU promoters might have several advantages for expression ofheterologous genes in plants. Some SSU promoters are very highlyexpressed and could give rise to expression levels as high or higherthan those observed with the CaMV35S promoter. The tissue distributionof expression from SSU promoters is different from that of the CaMV35Spromoter, so for control of some insect pests, it may be advantageous todirect the expression of crystal proteins to those cells in which SSU ismost highly expressed. For example, although relatively constitutive, inthe leaf the CaMV35S promoter is more highly expressed in vasculartissue than in some other parts of the leaf, while most SSU promotersare most highly expressed in the mesophyll cells of the leaf. Some SSUpromoters also are more highly tissue specific, so it could be possibleto utilize a specific SSU promoter to express the protein of the presentinvention in only a subset of plant tissues, if for example expressionof such a protein in certain cells was found to be deleterious to thosecells. For example, for control of Colorado potato beetle in potato, itmay be advantageous to use SSU promoters to direct crystal proteinexpression to the leaves but not to the edible tubers.

Utilizing SSU CTP sequences to localize crystal proteins to thechloroplast might also be advantageous. Localization of the B.thuringiensis crystal proteins to the chloroplast could protect thesefrom proteases found in the cytoplasm. This could stabilize the proteinsand lead to higher levels of accumulation of active toxin. cry genescontaining the CTP could be used in combination with the SSU promoter orwith other promoters such as CaMV35S.

5.36 Example 36 Targeting of Cry* Proteins to the Extracellular Space orVacuole Through the Use of Signal Peptides

The B. thuringiensis proteins produced from the synthetic genesdescribed here are localized to the cytoplasm of the plant cell, andthis cytoplasmic localization results in plants that are insecticidallyeffective. It may be advantageous for some purposes to direct the B.thuringiensis proteins to other compartments of the plant cell.Localizing B. thuringiensis proteins in compartments other than thecytoplasm may result in less exposure of the B. thuringiensis proteinsto cytoplasmic proteases leading to greater accumulation of the proteinyielding enhanced insecticidal activity. Extracellular localizationcould lead to more efficient exposure of certain insects to the B.thuringiensis proteins leading to greater efficacy. If a B.thuringiensis protein were found to be deleterious to plant cellfunction, then localization to a noncytoplasmic compartment couldprotect these cells from the protein.

In plants as well as other eukaryotes, proteins that are destined to belocalized either extracellularly or in several specific compartments aretypically synthesized with an N-terminal amino acid extension known asthe signal peptide. This signal peptide directs the protein to enter thecompartmentalization pathway, and it is typically cleaved from themature protein as an early step in compartmentalization. For anextracellular protein, the secretory pathway typically involvescotranslational insertion into the endoplasmic reticulum with cleavageof the signal peptide occurring at this stage. The mature protein thenpasses through the Golgi body into vesicles that fuse with the plasmamembrane thus releasing the protein into the extracellular space.Proteins destined for other compartments follow a similar pathway. Forexample, proteins that are destined for the endoplasmic reticulum or theGolgi body follow this scheme, but they are specifically retained in theappropriate compartment. In plants, some proteins are also targeted tothe vacuole, another membrane bound compartment in the cytoplasm of manyplant cells. Vacuole targeted proteins diverge from the above pathway atthe Golgi body where they enter vesicles that fuse with the vacuole.

A common feature of this protein targeting is the signal peptide thatinitiates the compartmentalization process. Fusing a signal peptide to aprotein will in many cases lead to the targeting of that protein to theendoplasmic reticulum. The efficiency of this step may depend on thesequence of the mature protein itself as well. The signals that direct aprotein to a specific compartment rather than to the extracellular spaceare not as clearly defined. It appears that many of the signals thatdirect the protein to specific compartments are contained within theamino acid sequence of the mature protein. This has been shown for somevacuole targeted proteins, but it is not yet possible to define thesesequences precisely. It appears that secretion into the extracellularspace is the “default” pathway for a protein that contains a signalsequence but no other compartmentalization signals. Thus, a strategy todirect B. thuringiensis proteins out of the cytoplasm is to fuse thegenes for synthetic B. thuringiensis genes to DNA sequences encodingknown plant signal peptides. These fusion genes will give rise to B.thuringiensis proteins that enter the secretory pathway, and lead toextracellular secretion or targeting to the vacuole or othercompartments. Signal sequences for several plant genes have beendescribed. One such sequence is for the tobacco pathogenesis relatedprotein PR1b has been previously described (Cornelissen et al., 1986).The PR1b protein is normally localized to the extracellular space.Another type of signal peptide is contained on seed storage proteins oflegumes. These proteins are localized to the protein body of seeds,which is a vacuole like compartment found in seeds. A signal peptide DNAsequence for the β-subunit of the 7S storage protein of common bean(Phaseolus vulgaris), PvuB has been described (Doyle et al., 1986).Based on the published these published sequences, genes may besynthesized chemically using oligonucleotides that encode the signalpeptides for PR1b and PvuB. In some cases to achieve secretion orcompartmentalization of heterologous proteins, it may be necessary toinclude some amino acid sequence beyond the normal cleavage site of thesignal peptide. This may be necessary to insure proper cleavage of thesignal peptide.

5.37 Example 37 Isolation of Transgenic Maize Resistant to Diabroticsspp. Using Cry3Bb Variants

5.37.1 Plant Gene Construction

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA. Transcription of DNA into mRNA is regulated by a region ofDNA usually referred to as the “promoter”. The promoter region containsa sequence of bases that signals RNA polymerase to associate with theDNA and to initiate the transcription of mRNA using one of the DNAstrands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., U.S. Pat. No. 5,463,175,specifically incorporated herein by reference).

The particular promoter selected should be capable of causing sufficientexpression of the enzyme coding sequence to result in the production ofan effective amount of protein. One set of preferred promoters areconstitutive promoters such as the CaMV35S or FMV35S promoters thatyield high levels of expression in most plant organs (U.S. Pat. No.5,378,619, specifically incorporated herein by reference). Another setof preferred promotors are root enhanced or specific promoters such asthe CaMV derived 4 as-1 promoter or the wheat POX1 promoter (U.S. Pat.No. 5,023,179, specifically incorporated herein by reference; Hertig etal., 1991). The root enhanced or specific promoters would beparticularly preferred for the control of corn rootworm (Diabroticusspp.) in transgenic corn plants.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence.

For optimized expression in monocotyledenous plants such as maize, anintron should also be included in the DNA expression construct. Thisintron would typically be placed near the 5′ end of the mRNA inuntranslated sequence. This intron could be obtained from, but notlimited to, a set of introns consisting of the maize hsp70 intron (U.S.Pat. No. 5,424,412; specifically incorporated herein by reference) orthe rice Actl intron (McElroy et al., 1990). As shown below, the maizehsp70 intron is useful in the present invention.

As noted above, the 3′ non-translated region of the chimeric plant genesof the present invention contains a polyadenylation signal whichfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9gene (Fischhoff et al., 1987).

5.37.2 Plant Transformation and Expression

A chimeric plant gene containing a structural coding sequence of thepresent invention can be inserted into the genome of a plant by anysuitable method. Suitable plant transformation vectors include thosederived from a Ti plasmid of Agrobacterium tumefaciens, as well as thosedisclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985)and Eur. Pat. Appl. Publ. No. EP0120516. In addition to planttransformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen (Fromm et al.,1986; Armstrong et al., 1990; Fromm et al., 1990).

5.37.3 Construction of Monocot Plant Expression Vectors for Cry3BbVariants

5.37.3.1 Design of Cry3Bb Variant Genes for Plant Expression

For efficient expression of the cry3Bb variants in transgenic plants,the gene encoding the variants must have a suitable sequence composition(Diehn et al, 1996). One example of such a sequence is shown for thev11231 gene (SEQ ID NO:99) which encodes the Cry3Bb11231 variant protein(SEQ ID NO:100) with Diabrotica activity. This gene was derived viamutagenesis (Kunkel, 1985) of a cry3Bb synthetic gene (SEQ ID NO:101)encoding a protein essentially homologous to the protein encoded by thenative cry3Bb gene (Gen Bank Accession Number m89794, SEQ ID NO:102).The following oligonucleotides were used in the mutagenesis of theoriginal cry3Bb synthetic gene (SEQ ID NO:101) to create the v11231 gene(SEQ ID NO:99):

Oligo #1: 5′-TAGGCCTCCATCCATGGCAAACCCTAACAATC-3′ (SEQ ID NO:103) Oligo#2: 5′-TCCCATCTTCCTACTTACGACCCTGCAGAAATACGGTCCAAC-3′ (SEQ ID NO:104)Oligo #3: 5′-GACCTCACCTACCAAACATTCGATCTTG-3′ (SEQ ID NO:105) Oligo #4:5′-CGAGTTCTACCGTAGGCAGCTCAAG-3′ (SEQ ID NO:106)5.37.3.2 Construction of Cry3Bb Monocot Plant Expression Vector

To place the cry3Bb variant gene v11231 in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and link to the hsp70intron followed by a nopaline synthase polyadenylation site as in U.S.Pat. No. 5,424,412, specifically incorporated herein by reference), thevector pMON19469 was digested with NcoI and EcoRI. The larger vectorband of approximately 4.6 kb was electrophoresed, purified, and ligatedwith T4 DNA ligase to the NcoI-EcoRI fragment of approximately 2 kbcontaining the v11231 gene (SEQ ID NO:99). The ligation mix wastransformed into E. coli, carbenicillin resistant colonies recovered andplasmid DNA recovered by DNA miniprep procedures. This DNA was subjectedto restriction endonuclease analysis with enzymes such as NcoI and EcoRI(together) NotI, and PstI to identify clones containing pMON33708 (thev11231 coding sequence fused to the hsp70 intron under control of theenhanced CaMV35S promoter).

To place the v11231 gene in a vector suitable for recovery of stablytransformed and insect resistant plants, the 3.75-kb NotI restrictionfragment from pMON33708 containing the lysine oxidase coding sequencefused to the hsp70 intron under control of the enhanced CaMV35S promoterwas isolated by gel electrophoresis and purification. This fragment wasligated with pMON30460 treated with NotI and calf intestinal alkalinephosphatase (pMON30460 contains the neomycin phosphotransferase codingsequence under control of the CaMV35S promoter). Kanamycin resistantcolonies were obtained by transformation of this ligation mix into E.coli and colonies containing pMON33710 identified by restrictionendonuclease digestion of plasmid miniprep DNAs. Restriction enzymessuch as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII can be used toidentify the appropriate clones containing the NotI fragment ofpMON33708 in the NotI site of pMON30460 (i.e. pMON33710) in theorientation such that both genes are in tandem (i.e. the 3′ end of thev11231 expression cassette is linked to the 5′ end of the nptIIexpression cassette). Expression of the v11231 protein by pMON33710 incorn protoplasts was confirmed by electroporation of pMON33710 DNA intoprotoplasts followed by protein blot and ELISA analysis. This vector canbe introduced into the genomic DNA of corn embryos by particle gunbombardment followed by paromomycin selection to obtain corn plantsexpressing the v11231 gene essentially as described in U.S. Pat. No.5,424,412, specifically incorporated herein by reference.

In this example, the vector was introduced via cobombardment with ahygromycin resistance conferring plasmid into immature embryo scutella(IES) of maize, followed by hygromycin selection, and regeneration.Transgenic corn lines expressing the v11231 protein were identified byELISA analysis. Progeny seed from these events were subsequently testedfor protection from Diabrotica feeding.

5.37.3.3 In Planta Performance of Cry3Bb.11231

Transformed corn plants expressing Cry3Bb.11231 protein were challengedwith western corn rootworm (WCR) larvae in both a seedling and 10 inchpot assay. The transformed genotype was A634, where the progeny of theR0 cross by A634 was evaluated. Observations included effect on larvaldevelopment (weight), root damage rating (RDR), and protein expression.The transformation vector containing the cry3Bb gene was pMON33710.Treatments included the positive and negative iso-populations for eachevent and an A634 check.

The seedling assay consisted of the following steps: (i) single seedswere placed in 1 oz cups containing potting soil; (ii) at spiking, eachseedling was infested with 4 neonate larvae; and (iii) afterinfestation, seedlings were incubated for 7 days at 25° C., 50% RH, and14:10 (L:D) photo period. Adequate moisture was added to the pottingsoil during the incubation period to maintain seedling vigor.

The 10 inch pot assay consisted of the following steps: (i) single seedswere placed in 10 inch pots containing potting soil; (ii) at 14 dayspost planting, each pot was infested with 800 eggs which have beenpre-incubated such that hatch would occur 5-7 days post infestation; and(iii) after infestation, plants were incubated for 4 weeks under thesame environmental conditions as the seedling assay. Pots were both suband top irrigated daily.

For the seedling assay, on day 7 plants were given a root damage rating,and surviving larvae were weighed. Also at this time, Cry3Bb proteinconcentrations in the roots were determined by ELISA. The scale used forthe seedling assay to assess root damage is as follows: RDR (root damagerating) 0=no visible feeding; RDR 1=very light feeding; RDR 2=lightfeeding; RDR 3=moderate feeding; RDR 4=heavy feeding; and RDR 5=veryheavy feeding.

Results of the seedling assay are shown in Table 26. Plants expressingCry3Bb protein were completely protected by WCR feeding, where survivinglarvae within this treatment had not grown. Mean larval weights rangedfrom 2.03-2.73 mg for the nonexpressing treatments, where the survivinglarval average weight was 0.11 mg on the expressing cry3Bb treatment.Root damage ratings were 3.86 and 0.33 for the nonexpressing andexpressing isopopulations, respectively. Larval survival ranged from75-85% for the negative and check treatments, where only 25% of thelarvae survived on the Cry3Bb treatment.

TABLE 26 EFFECT OF CRY3BB EXPRESSING PLANTS ON WCR LARVAE IN A SEEDLINGASSAY Plants Larvae Root % Mean ± SD Event Treatment N (ppm) RDR ± SD NSurv Wt. (mg) 16 Negative 7 0.0 3.86 ± 0.65 21 75 2.73 ± 1.67 16Positive 3 29.01 0.33 ± 0.45  3 25 0.11 ± 0.07 A634 Check 4 0.0 — 13 812.03 ± 0.83

For the 10 inch pot assay, at 4 weeks post infestation plant height wasrecorded and a root damage rating (Iowa 1-6 scale; Hills and Peters,1971) was given.

Results of the 10 inch pot assay are shown in Table 27. Plantsexpressing Cry3Bb protein had significantly less feeding damage and weretaller than the non-expressing plants. Event 16, the higher of the twoexpressing events provided nearly complete control. The negativetreatments had very high root damage ratings indicating very high insectpressure. The positive mean root damage ratings were 3.4 and 2.2 forevent 6 and 16, respectively. Mean RDR for the negative treatment was5.0 and 5.6.

TABLE 27 EFFECT OF CRY3BB EXPRESSING CORN IN CONTROLLING WCR LARVALFEEDING IN A 10 INCH POT ASSAY Root Plant Event Treatment N (ppm) RDR ±SD Height (cm)  6 Negative 7 0.0 5.0 ± 1.41 49.7 ± 18.72  6 Positive 57.0 3.4 ± 1.14 73.9 ± 8.67 16 Negative 5 0.0 5.6 ± 0.89 61.2 ± 7.75 16Positive 5 55.0 2.2 ± 0.84 83.8 ± 7.15

In summary, corn plants expressing Cry3Bb protein have a significantbiological effect on WCR larval development as seen in the seedlingassay. When challenged with very high infestation levels, plantsexpressing the Cry3Bb protein were protected from WCR larval feedingdamage as illustrated in the 10 inch pot assay.

6.0 Brief Description of the Sequence Identifiers

-   -   SEQ ID NO:1 DNA sequence of cry3Bb.11221 gene.    -   SEQ ID NO:2 Amino acid sequence of Cry3Bb.11221 polypeptide.    -   SEQ ID NO:3 DNA sequence of cry3Bb.11222 gene.    -   SEQ ID NO:4 Amino acid sequence of Cry3Bb.11222 polypeptide.    -   SEQ ID NO:5 DNA sequence of cry3Bb.11223 gene.    -   SEQ ID NO:6 Amino acid sequence of Cry3Bb.11223 polypeptide.    -   SEQ ID NO:7 DNA sequence of cry3Bb.11224 gene.    -   SEQ ID NO:8 Amino acid sequence of Cry3Bb.11224 polypeptide.    -   SEQ ID NO:9 DNA sequence of cry3Bb.11225 gene.    -   SEQ ID NO:10 Amino acid sequence of Cry3Bb.11225 polypeptide.    -   SEQ ID NO:11 DNA sequence of cry3Bb.11226 gene.    -   SEQ ID NO:12 Amino acid sequence of Cry3Bb.11226 polypeptide.    -   SEQ ID NO:13 DNA sequence of cry3Bb.11227 gene.    -   SEQ ID NO:14 Amino acid sequence of Cry3Bb.11227 polypeptide.    -   SEQ ID NO:15 DNA sequence of cry3Bb.11228 gene.    -   SEQ ID NO:16 Amino acid sequence of Cry3Bb.11228 polypeptide.    -   SEQ ID NO:17 DNA sequence of cry3Bb.11229 gene.    -   SEQ ID NO:18 Amino acid sequence of Cry3Bb.11229 polypeptide.    -   SEQ ID NO:19 DNA sequence of cry3Bb.11230 gene.    -   SEQ ID NO:20 Amino acid sequence of Cry3Bb.11230 polypeptide.    -   SEQ ID NO:21 DNA sequence of cry3Bb.11231 gene.    -   SEQ ID NO:22 Amino acid sequence of Cry3Bb.11231 polypeptide.    -   SEQ ID NO:23 DNA sequence of cry3Bb.11232 gene.    -   SEQ ID NO:24 Amino acid sequence of Cry3Bb.11232 polypeptide.    -   SEQ ID NO:25 DNA sequence of cry3Bb.11233 gene.    -   SEQ ID NO:26 Amino acid sequence of Cry3Bb.11233 polypeptide.    -   SEQ ID NO:27 DNA sequence of cry3Bb.1234 gene.    -   SEQ ID NO:28 Amino acid sequence of Cry3Bb.11234 polypeptide.    -   SEQ ID NO:29 DNA sequence of cry3Bb.11235 gene.    -   SEQ ID NO:30 Amino acid sequence of Cry3Bb.11235 polypeptide.    -   SEQ ID NO:31 DNA sequence of cry3Bb.11236 gene.    -   SEQ ID NO:32 Amino acid sequence of Cry3Bb.11236 polypeptide.    -   SEQ ID NO:33 DNA sequence of cry3Bb.11237 gene.    -   SEQ ID NO:34 Amino acid sequence of Cry3Bb.11237 polypeptide.    -   SEQ ID NO:35 DNA sequence of cry3Bb.11238 gene.    -   SEQ ID NO:36 Amino acid sequence of Cry3Bb.11238 polypeptide.    -   SEQ ID NO:37 DNA sequence of cry3Bb.11239 gene.    -   SEQ ID NO:38 Amino acid sequence of Cry3Bb.11239 polypeptide.    -   SEQ ID NO:39 DNA sequence of cry3Bb.11241 gene.    -   SEQ ID NO:40 Amino acid sequence of Cry3Bb.11241 polypeptide.    -   SEQ ID NO:41 DNA sequence of cry3Bb.11242 gene.    -   SEQ ID NO:42 Amino acid sequence of Cry3Bb.11242 polypeptide.    -   SEQ ID NO:43 DNA sequence of cry3Bb.11032 gene.    -   SEQ ID NO:44 Amino acid sequence of Cry3Bb.11032 polypeptide.    -   SEQ ID NO:45 DNA sequence of cry3Bb.11035 gene.    -   SEQ ID NO:46 Amino acid sequence of Cry3Bb.11035 polypeptide.    -   SEQ ID NO:47 DNA sequence of cry3Bb.11036 gene.    -   SEQ ID NO:48 Amino acid sequence of Cry3Bb.11036 polypeptide.    -   SEQ ID NO:49 DNA sequence of cry3Bb.11046 gene.    -   SEQ ID NO:50 Amino acid sequence of Cry3Bb.11046 polypeptide.    -   SEQ ID NO:51 DNA sequence of cry3Bb.11048 gene.    -   SEQ ID NO:52 Amino acid sequence of Cry3Bb.11048 polypeptide.    -   SEQ ID NO:53 DNA sequence of cry3Bb.11051 gene.    -   SEQ ID NO:54 Amino acid sequence of Cry3Bb.11051 polypeptide.    -   SEQ ID NO:55 DNA sequence of cry3Bb.11057 gene.    -   SEQ ID NO:56 Amino acid sequence of Cry3Bb.11057 polypeptide.    -   SEQ ID NO:57 DNA sequence of cry3Bb.11058 gene.    -   SEQ ID NO:58 Amino acid sequence of Cry3Bb.11058 polypeptide.    -   SEQ ID NO:59 DNA sequence of cry3Bb.11081 gene.    -   SEQ ID NO:60 Amino acid sequence of Cry3Bb.11081 polypeptide.    -   SEQ ID NO:61 DNA sequence of cry3Bb.11082 gene.    -   SEQ ID NO:62 Amino acid sequence of Cry3Bb.11082 polypeptide.    -   SEQ ID NO:63 DNA sequence of cry3Bb.11083 gene.    -   SEQ ID NO:64 Amino acid sequence of Cry3Bb.11083 polypeptide.    -   SEQ ID NO:65 DNA sequence of cry3Bb.11084 gene.    -   SEQ ID NO:66 Amino acid sequence of Cry3Bb.11084 polypeptide.    -   SEQ ID NO:67 DNA sequence of cry3Bb.11095 gene.    -   SEQ ID NO:68 Amino acid sequence of Cry3Bb.11095 polypeptide.    -   SEQ ID NO:69 DNA sequence of cry3Bb.60 gene.    -   SEQ ID NO:70 Amino acid sequence of Cry3Bb.60 polypeptide.    -   SEQ ID NO:71 Primer FW001.    -   SEQ ID NO:72 Primer FW006.    -   SEQ ID NO:73 Primer MVT095.    -   SEQ ID NO:74 Primer MVT097.    -   SEQ ID NO:75 Primer MVT091.    -   SEQ ID NO:76 Primer MVT075.    -   SEQ ID NO:77 Primer MVT076.    -   SEQ ID NO:78 Primer MVT111.    -   SEQ ID NO:79 Primer MVT094.    -   SEQ ID NO:80 Primer MVT103.    -   SEQ ID NO:81 Primer MVT081.    -   SEQ ID NO:82 Primer MVT085.    -   SEQ ID NO:83 Primer A.    -   SEQ ID NO:84 Primer B.    -   SEQ ID NO:85 Primer C.    -   SEQ ID NO:86 Primer D.    -   SEQ ID NO:87 Primer E.    -   SEQ ID NO:88 Primer F.    -   SEQ ID NO:89 Primer G.    -   SEQ ID NO:90 Primer WD112.    -   SEQ ID NO:91 Primer WD115.    -   SEQ ID NO:92 Primer MVT105.    -   SEQ ID NO:93 Primer MVT092.    -   SEQ ID NO:94 Primer MVT070.    -   SEQ ID NO:95 Primer MVT083.    -   SEQ ID NO:96 N-terminal amino acid of Cry3Bb polypeptide.    -   SEQ ID NO:97 DNA sequence of wild-type cry3Bb gene.    -   SEQ ID NO:98 Amino acid sequence of wild-type Cry3Bb        polypeptide.    -   SEQ ID NO:99 Plantized DNA sequence for cry3Bb.11231 gene.    -   SEQ ID NO:100 Amino acid sequence of plantized Cry3Bb.11231        polypeptide.    -   SEQ ID NO:101 DNA sequence of cry3Bb gene used to prepare SEQ ID        NO:99.    -   SEQ ID NO:102 DNA sequence of wild-type cry3Bb gene, Genbank        #M89794.    -   SEQ ID NO:103 DNA sequence of Oligo #1.    -   SEQ ID NO:104 DNA sequence of Oligo #2.    -   SEQ ID NO:105 DNA sequence of Oligo #3.    -   SEQ ID NO:106 DNA sequence of Oligo #4.    -   SEQ ID NO:107 DNA sequence of cry3Bb.11098 gene.    -   SEQ ID NO:108 Amino acid sequence of Cry3Bb.11098 polypeptide.        7.0 References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A transgenic plant comprising a gene encoding a modified Cry3Bb*polypeptide, wherein said modified polypeptide comprises one or moreamino acids within loop β1,α8 replaced with one or more amino acidshaving increased hydrophobicity, wherein said replacement results in oneor more amino acid substitutions selected from the group consisting ofSer311 replaced by leucine, Asn313 replaced by threonine, and Glu317replaced by lysine, wherein said modified Cry3Bb* polypeptide is SEQ IDNO:60, SEQ ID NO:66, SEQ ID NO:108, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:22, SEQ ID NO:100, SEQ ID NO:24 or SEQ ID NO:30.
 2. The transgenicplant of claim 1, wherein said modified polypeptide further comprisesamino acid substitution of His231 replaced by arginine, wherein saidmodified Cry3Bb* polypeptide is SEQ ID NO:108, SEQ ID NO:22, SEQ IDNO:100 or SEQ ID NO:30.
 3. The transgenic plant of claim 2, wherein saidmodified Cry3Bb* polypeptide is SEQ ID NO:100.
 4. The transgenic plantof claim 2, wherein said modified polypeptide further comprises aminoacid substitution of Gin348 replaced by arginine.
 5. A progeny plant orseed from the transgenic plant of claim 4, 1, 2 or 3, wherein saidprogeny plant or seed comprises said gene encoding said modified Cry3Bb*polypeptide.
 6. A seed from the progeny plant of claim 5, wherein saidseed comprises said gene encoding said modified Cry3Bb* polypeptide. 7.A plant from the seed of claim 5 or 2, wherein said plant comprises saidgene encoding said modified Cry3Bb* polypeptide.
 8. A plant from theseed of claim 6, wherein said plant comprises said gene encoding saidmodified Cry3Bb* polypeptide.
 9. A method of preparing aColeopteran-resistant transgenic plant, wherein the method comprises thesteps of: (a) obtaining a nucleic acid segment comprising a geneencoding a modified Cry3Bb* polypeptide, wherein: said modifiedpolypeptide comprises one or more point mutations in or near α helix 4,wherein said one or more point mutations result in at least one aminoacid substitution of Asp165 to Gly; (b) transforming a plant cell withsaid nucleic acid segment; and (c) regenerating from said plant cell atransgenic plant, which expresses said modified Cry3Bb* polypeptide andwherein said transgenic plant is resistant to Coleopteran insects ascompared to a non-transformed plant and wherein the transgenic plant isresistant to corn rootworm insects as compared to a non-transformedplant.
 10. The method of claim 9, wherein step a) further comprisesoperatively linking the gene to a promoter, and introducing said nucleicacid segment into a vector, and wherein step b) comprises transforming aplant cell with said vector.
 11. The method of claim 9, wherein saidmodified Cry3Bb* polypeptide is SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:50, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66 or SEQ IDNO:108.
 12. The method of claim 9, wherein said modified polypeptidefurther comprises one or more of the amino acid substitutions selectedfrom the group consisting of His231 replaced by arginine, Ser311replaced by leucine, Asn313 replaced by threonine, Glu317 replaced bylysine, and Gln348 replaced by arginine, wherein said modifiedpolypeptide is SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66 or SEQ IDNO:108.